[go: up one dir, main page]

AU2013320165A1 - A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma - Google Patents

A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma Download PDF

Info

Publication number
AU2013320165A1
AU2013320165A1 AU2013320165A AU2013320165A AU2013320165A1 AU 2013320165 A1 AU2013320165 A1 AU 2013320165A1 AU 2013320165 A AU2013320165 A AU 2013320165A AU 2013320165 A AU2013320165 A AU 2013320165A AU 2013320165 A1 AU2013320165 A1 AU 2013320165A1
Authority
AU
Australia
Prior art keywords
sample
expression level
hca
act
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
AU2013320165A
Inventor
Aurelien De Reynies
Pierre Laurent-Puig
Jean-Charles NAULT
Jessica Zucman-Rossi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
IntegraGen SA
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
IntegraGen SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Descartes, IntegraGen SA filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of AU2013320165A1 publication Critical patent/AU2013320165A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medical Informatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Evolutionary Biology (AREA)
  • Theoretical Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Data Mining & Analysis (AREA)
  • Databases & Information Systems (AREA)
  • Epidemiology (AREA)

Abstract

The present invention relates to the technical field of liver diseases, their classification and diagnosis. It provides a new method for classifying a liver sample between non- hepatocellular sample; hepatocellular carcinoma (HCC) sample with further classification into one of subgroups G1 to G6; focal nodule dysplasia (FNH) sample; hepatocellular adenoma (HCA) sample with further classification into HNF1A mutated HCA, inflammatory HCA, β catenin mutated HCA or other HCA sample; and other benign liver sample, based on determination in vitro of genes expression profiles and analysis of the expression profile using algorithms calibrated with reference samples. The invention also provides kits for the classification of liver samples, and methods of treatment of liver disease in a subject based on a preliminary classification of a liver sample of said subject.

Description

WO 2014/044853 PCT/EP2013/069751 1 A NEW METHOD FOR CLASSIFICATION OF LIVER SAMPLES AND DIAGNOSIS OF FOCAL NODULE DYSPLASIA, HEPATOCELLULAR ADENOMA, AND HEPATOCELLULAR CARCINOMA 5 TECHNICAL FIELD OF THE INVENTION The present invention relates to the technical field of liver diseases, their classification and diagnosis. It provides a new method for classifying a liver sample between non hepatocellular sample; hepatocellular carcinoma (HCC) sample with further classification into one of subgroups G1 to G6; focal nodule dysplasia (FNH) sample; 10 hepatocellular adenoma (HCA) sample with further classification into HNF1A mutated HCA, inflammatory HCA, P catenin mutated HCA or other HCA sample; and other benign liver sample, based on determination in vitro of genes expression profiles and analysis of the expression profile using algorithms calibrated with reference samples. The invention also provides kits for the classification of liver samples, and methods of 15 treatment of liver disease in a subject based on a preliminary classification of a liver sample of said subject. BACKGROUND ART Hepatocellular carcinoma (HCC) represents one of the leading worldwide causes of death by cancer (El Serag H NEJM 2011). Despite the widespread use of imaging/non 20 invasive criteria for the diagnosis of HCC developed on cirrhosis, the differential diagnosis between HCC and others liver tumors remains difficult, even for an expert pathologist (international consensus group 2009). In this setting, regenerative and dysplastic macronodule, cholangiocarcinoma or metastasis of cancers of other tissue origin constitute classical pitfalls (Forner A Lancet 2012). Moreover, non-invasive 25 criteria have not been validated for the diagnosis of HCC developed in non-cirrhotic liver contributing for 10 % of the cases in western countries and more than 20 % in eastern countries (Forner A Hepatology 2008). In this setting, tumor biopsy is mandatory and differential diagnosis with benign hepatocellular tumors (focal nodular hyperplasia, FNH and hepatocellular adenoma, HCA) could be challenging, especially 30 between very well differentiated HCC and HCA (Bioulac-Sage P, sem liv dis 2011). Moreover, HCA constitute a heterogeneous group of benign liver tumors and a genotype/phenotype classification related to prognosis was recently identified (Zucman Rossi J Hepatology 2006; Van aalten SM J hepatol 2011). Four groups of HCA (HNF1A mutated, P catenin mutated, inflammatory and unclassified hepatocellular 35 adenomas) were described and HCA with mutation activating P catenin was associated with an increased risk of malignant transformation in HCC.
WO 2014/044853 2 PCT/EP2013/069751 Therefore, benign and malignant hepatocellular tumors comprise various subgroups of tumors defined by specific phenotypic and molecular features, which leads to diagnosis pitfalls and difficulty to assess their prognosis. There is thus a need for new tools that help clinicians and pathologists in clinical 5 practice for reliably distinguishing between the various types of tissues that can be present in a liver sample (hepatocellular or not; if hepatocellular, benign or malignant; if benign hepatocellular, focal nodule hyperplasia, hepatocellular adenoma, or none of both; if hepatocellular adenoma, which type of it), and thus to reliably classify liver samples taken from subjects suspected to suffer from a liver tumor. 10 Indeed, depending on the classification of the liver sample and thus on the final diagnosis, the patient will not be given the same treatment: - In case of benign focal nodule hyperplasia (FNH), therapeutic abstention without follow up is recommended; - In case of benign hepatocellular adenoma (HCA), usual treatments include 15 surgical resection or therapeutic abstention with follow up. The selection of the best treatment may also depend on the more precise classification of HCA into HNF1A mutated, inflammatory, and P catenin mutated HCA. For instance, if the sample is diagnosed as HNF1A mutated HCA smaller than 5 cm, a follow up with imaging/clinical follow up only may be particularly useful, because of the 20 low risk of hemorrhage and malignant transformation. If the sample is diagnosed as HNF1A mutated HCA with a size of more than 5 cm, a treatment with surgical resection may be particularly useful, because of the risk of hemorrhage. If the sample is diagnosed as inflammatory HCA with a size of less than 5 cm then a follow up with imaging/clinical follow up only may be 25 particularly useful, because of the low risk of hemorrhage and malignant transformation. If the sample is diagnosed as Inflammatory HCA with a size of more than 5 cm, then a treatment with surgical resection, may be particularly useful, because of the risk of hemorrhage. If the sample is diagnosed as P catenin mutated HCA whatever the size, then a curative treatment with surgical 30 resection may be particularly useful, because of the high risk of malignant transformation. - In case of hepatocellular carcinoma (HCC), the first treatment generally consists in tumor surgical resection, although alternative treatment may be used if tumor surgical resection is not possible. In addition, various adjuvant 35 therapies may be administered after tumor surgical resection. Such adjuvant therapies include cytotoxic chemotherapy (in particular doxorubicin or association of gemcitabine and oxaliplatine) and/or targeted therapy (in particular sorafenib). The selection of the best treatment strategy (including the use or not of adjuvant therapy) may depend on the more precise type of HCC 40 (see classification of HCC into one of subgroups G1 to G6 described in W02007/063118A1) and/or on the prognosis of the patient. In particular, in WO 2014/044853 3 PCT/EP2013/069751 case of bad prognosis, adjuvant therapy is generally given, while it is not systematically the case if the prognosis is good. In addition, if the liver sample has been further classified as HCC subgroup G1, then a treatment with IGFR1 inhibitor may be particularly useful, because of the activation of insulin growth 5 factor pathway. If the liver sample has been further classified as HCC subgroup G1 or G2, then a treatment with Akt/mtor inhibitor may be particularly useful, because the activation of akt/mtor pathway. If the liver sample has been further classified as HCC subgroup G3, then a treatment with proteasome inhibitor may be particularly useful, because of the dysregulation of cell/cycle genes. If the 10 liver sample has been further classified as HCC subgroup G5 or G6, then a treatment with Wnt inhibitor may be particularly useful, because of activation of Wnt/catenin pathway. In this setting, a simple classification/diagnosis tool based on molecular profiling of a subject's liver sample would be very helpful. 15 Several genes have been associated to the classification of liver samples or the diagnosis of particular liver diseases. For instance, genes differentially expressed in hepatocellular and non-hepatocellular tissue have been described in Odom et al-2004. Genes associated to benign or malignant hepatocellular tumors have been identified in Llovet et al-2006, Capurro et al-2003, Chuma et al-2003, Tsunedomi et al-2005 and 20 Kondoh et al-1999. Genes differentially expressed in focal nodule hyperplasia (FNH) have been disclosed in Rebouissou et al-2008 and Paradis et al-2003. Genes differentially expressed in HNF1A mutated HCA have been disclosed in Rebouissou et al-2007 and Bioulac Sage et al-2007. Genes associated to P catenin mutations have been described in Boyault et al-2007, Bioulac Sage et al-2007, Cadoret et al-2002, 25 Yamamoto et al-2005, Benhamouche et al-2006, and Rebouissou et al-2008. Genes differentially expressed in inflammatory HCA have been disclosed in Rebouissou et al 2009 and Bioulac Sage et al-2007. However, there has been no disclosure in the prior art of a true method permitting to simply and reliably classify a liver sample among the various types of liver diseases, 30 and to simply and reliably diagnose the presence of non-hepatocellular tissue in liver, malign hepatocellular carcinoma (HCC), benign focal nodule hyperplasia (FNH), hepatocellular adenoma and its subtypes. Based on a new strategy of analysis of microarray and quantitative PCR data obtained from various types of liver samples, the inventors have constructed a simple and 35 reliable molecular algorithm for the precise classification and diagnosis of liver samples. In particular, the inventors have established several signatures able: * To reliably distinguish between hepatocellular and non-hepatocellular sample (metastasis of other tissue origin, cholangiocarcinoma), or between benign and malignant (hepatocellular carcinoma) hepatocellular samples; 40 0 To precisely diagnose, among benign hepatocellular samples the presence of focal nodule hyperplasia (FNH) or hepatocellular adenoma (HCA); and WO 2014/044853 PCT/EP2013/069751 To precisely diagnose, among HCA samples, the type of HCA sample: HNF1A mutated HCA, inflammatory HCA, P catenin mutated HCA, or other HCA. A global set of 55 genes permits to reliably classify a liver between all those types of liver samples. 5 DESCRIPTION OF THE INVENTION The present invention thus relates to a method for classifying in vitro a liver sample as a non-hepatocellular sample, a hepatocellular carcinoma (HCC) sample, a focal nodule dysplasia (FNH) sample, a hepatocellular adenoma (HCA) sample or another benign liver sample, comprising: 10 a) Determining in vitro from said liver sample an expression profile comprising or consisting of the 38 following genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, 15 ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; b) Determining if said liver sample is a hepatocellular or a non-hepatocellular sample, based on the expression levels measured for an expression profile comprising or consisting of the 9 following genes: EPCAM, HNF4A, CYP3A7, 20 FABP1, HAL, AFP, GNMT, TFRC, and C8A, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; c) If said liver sample is a hepatocellular sample, then determining if said hepatocellular sample is a HCC sample or a benign hepatocellular sample, 25 based on the expression levels measured for an expression profile comprising or consisting of the 9 following genes: AFP, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, and ADM, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; 30 d) If said liver sample is a benign hepatocellular sample, then determining if said benign hepatocellular sample is a FNH sample, based on the expression levels measured for an expression profile comprising or consisting of the 13 following genes: HAL, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, and GIMAP5, and optionally one 35 or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; e) If said liver sample is a benign hepatocellular sample, then determining if said benign hepatocellular sample is a HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 13 40 following genes: HAL, CYP3A7, LCAT, LYVE1, AKR1B10, GLS2, KRT19, WO 2014/044853 5 PCT/EP2013/069751 ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; f) If said benign hepatocellular sample is neither a FNH sample nor a HCA 5 sample, then it is classified as another benign liver sample. In an advantageous embodiment, the method according to the invention further comprises, if the liver sample is diagnosed as a HCA sample, classifying said HCA sample into one of the following HCA subgroups: HNF1A mutated HCA, inflammatory 10 HCA, P catenin mutated HCA or other HCA, by: a) Further determining in vitro from said HCA sample an expression profile comprising or consisting of the 8 additional following genes: HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3; b) Determining if said HCA sample is or not a HNF1A mutated HCA sample, 15 based on the expression levels measured for an expression profile comprising or consisting of the 4 following genes: FABP1, ANGPT2, DHRS2, and UGT2B7, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; 20 c) Determining if said HCA sample is or not an inflammatory HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 7 following genes: ANGPT2, GLS2, EPHA1, CC15, HAMP, SAA2, and NRCAM, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated 25 with at least one reference liver sample; d) Determining if said HCA sample is or not a P catenin mutated HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 13 following genes: TFRC, HAL, CAP2, GLUL, HMGB3, LGR5, GIMAP5, AKR1B10, REG3A, AMACR, TAF9, LAPTM4B, and 30 IGF2BP3, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof, using at least one algorithm calibrated with at least one reference liver sample; e) If said HCA sample is neither a HNF1A mutated HCA sample, an inflammatory HCA sample, nor a P catenin mutated HCA sample, then it is classified as 35 another HCA sample. In another advantageous embodiment, the method according to the invention further comprises, if the liver sample is diagnosed as a HCC sample, classifying said HCC sample into one of subgroups G1 to G6 defined by the clinical and genetic main 40 features described in following Table 1: WO 2014/044853 6 PCT/EP2013/069751 G1 G2 G3 G4 G5 G6 Chromosome instability + + + - - Early relapse and death + + + - - TP53 mutation - + + - - HBV infection + + - - - Low copy number + - - - - High copy number - + - - - CTNNB1 mutation - - - - + + Satellite nodules - - - - - + wherein classification is made by: a) Further determining in vitro from said HCC sample an expression profile comprising or consisting of the 11 additional following genes: RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and SAE1; and 5 b) calculating 6 subgroup distances based on the expression levels measured for an expression profile comprising or consisting of the 16 following genes: RAB1A, REG3A, NRAS, RAMP3, MERTK, PIR, EPHA1, LAMA3, GOS2, HN1, PAK2, AFP, CYP2C9, CDH2, HAMP, and SAE1, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; and 10 c) classifying said HCC tumor in the subgroup for which the subgroup distance is the lowest. Such classification of HCC samples into subgroups G1 to G6 has already been described in detailed in W02007/063118A1, which content concerning such 15 classification is herein incorporated by reference. In a preferred embodiment, the HCC sample is classified into one of subgroups G1 to G6 using the following formula for calculating the distance of said HCC sample to each subgroup Gk, 15k6: 20 Distance (HCC sample, subgroup Gk) (ACt (HCC sample,subgroup Gk,genet) - p (subgroup Gk, genet)) 2 t=1..16 ofgenet) wherein for each genet and subgroup Gk, the p(subgroup Gk, genet) and o-(genet) values are the following: p G1 G2 G3 G4 G5 G6 o gene 1 (RABIA) -16.39 -16.04 -16.29 -17.15 -17.33 -16.95 0.23 gene 2 (PAP) -28.75 -27.02 -23.48 -27.87 -19.23 -11.33 16.63 gene 3 (NRAS) -16.92 -17.41 -16.25 -17.31 -16.96 -17.26 0.27 gene 4 (RAMP3) -23.54 -23.12 -25.34 -22.36 -23.09 -23.06 1.23 gene 5 (MERTK) -18.72 -18.43 -21.24 -18.29 -17.03 -16.16 7.23 gene 6 (PIR) -18.44 -19.81 -16.73 -18.28 -17.09 -17.25 0.48 WO 2014/044853 PCT/EP2013/069751 gene 7 (EPHAl) -16.68 -16.51 -19.89 -17.04 -18.70 -21.98 1.57 gene 8 (LAMA3) -20.58 -20.44 -20.19 -21.99 -18.77 -16.85 2.55 gene 9 (GOS2) -14.82 -17.45 -18.18 -14.78 -17.99 -16.06 3.88 gene 10 (HNI) -16.92 -17.16 -15.91 -17.88 -17.72 -17.93 0.54 gene 11 (PAK2) -17.86 -16.56 -16.99 -18.14 -17.92 -17.97 0.58 gene 12 (AFP) -16.68 -12.36 -26.80 -27.28 -25.97 -23.47 14.80 gene 13 (CYP2C9) -18.27 -16.99 -16.26 -16.23 -13.27 -14.44 5.47 gene 14 (CDH2) -15.20 -14.76 -18.91 -15.60 -15.48 -17.32 10.59 gene 15 (HAMP) -19.53 -20.19 -21.32 -18.51 -25.06 -26.10 13.08 gene 16 (SAEI) -17.37 -17.10 -16.79 -18.22 -17.72 -18.16 0.31 In the above methods according to the invention, when a HCC sample is further classified into one of subgroups G1 to G6, or when a HCA sample is further classified as a HNF1A mutated HCA sample, an inflammatory HCA sample, or a P catenin 5 mutated HCA sample, the two steps of determining in vitro the first expression profile for general classification and the second expression profile for further subgroup classification may be performed either simultaneously as only one step, or separately as two distinct steps. Preferably, they are performed simultaneously as only one step, since this is the simplest manner to do it. 10 In the above methods according to the invention, reference samples are used in order to calibrate an algorithm or a distance function, which may then be used to classify a new liver sample. In advantageous embodiments of the methods of the invention, reference samples used for calibrating algorithms or the distance function used for 15 interpreting expression profiles are the following: a) For determining if a liver sample is or not a hepatocellular sample: at least one (preferably several) hepatocellular sample and at least one (preferably several) non-hepatocellular sample; b) For determining if a hepatocellular sample is or not a HCC sample: at least 20 one (preferably several) benign sample and at least one (preferably several) HCC sample; c) For determining if a benign hepatocellular sample is or not a FNH sample: at least one (preferably several) FNH sample and at least one (preferably several) non-FNH benign hepatocellular sample; 25 d) For determining if a benign hepatocellular sample is or not a HCA sample: at least one (preferably several) HCA sample and at least one (preferably several) non-HCA benign hepatocellular sample; e) For determining if a HCA sample is or not a HNF1A mutated HCA sample: at least one (preferably several) HNF1A mutated HCA sample and at least one 30 (preferably several) non-HNF1A mutated HCA sample; WO 2014/044853 8 PCT/EP2013/069751 f) For determining if a HCA sample is or not an inflammatory HCA sample: at least one (preferably several) inflammatory HCA sample and at least one (preferably several) non-inflammatory HCA sample; g) For determining if a HCA sample is or not a P catenin mutated HCA sample: at 5 least one (preferably several) P catenin mutated HCA sample and at least one (preferably several) non-P catenin mutated HCA sample; and h) For classifying a HCC sample into one of subgroups G1 to G6: at least one (preferably several) sample of each G1 to G6 subgroups. 10 By "subject", it is meant any human subject, regardless of sex or age. By "liver sample", it is meant any sample obtained by taking part of the liver of a subject. By "hepatocellular" liver sample, it is intended to mean that the liver sample analyzed is mainly made of hepatocytes or progenitors of hepatocytes, which may or not be transformed. Conversely, by "non-hepatocellular" liver sample, it is intended to 15 mean that the liver sample is mainly made of cells others than hepatocytes or progenitors of hepatocytes. Non-hepatocellular liver samples notably include liver samples mainly made of metastases of cancers of non-hepatocellular origin (such as lung, breast, colon, or skin cancer for instance) and liver samples mainly made of cholangiocarcinoma, a cancer composed of mutated epithelial cells (or cells showing 20 characteristics of epithelial differentiation) that originate in the bile ducts which drain bile from the liver into the small intestine. Cholangiocarcinoma thus occurs in the liver but is made of non-hepatocellular cells. By "malignant hepatocellular samples", "hepatocellular carcinoma" or "HCC", it is intended to mean a primary malignancy of liver hepatocytes or hepatocytes 25 progenitors. HCC is generally diagnosed by histological analysis, and is characterized by hepatocytes proliferation with an elevated nuclear to cytoplasmic ratio, trabecular architecture and atypical nuclei. Benign hepatocellular samples include samples affected by FNH or HCA, and other benign hepatocellular samples. By "focal nodule hyperplasia" or "FNH", it is intended to 30 mean a benign tumor of the liver generally characterized by a central stellate scar seen in 60-70% of cases. Microscopically, a lobular proliferation of bland-appearing hepatocytes with a bile ductular proliferation and malformed vessels within the fibrous scar is the most common pattern. Other patterns include telangiectatic, hyperplastic adenomatous, and lesions with focal large-cell dysplasia. It is generally diagnosed by 35 histological analysis. By "hepatocellular adenoma", "hepatic adenoma", "hepadenoma" or "HCA", it is intended to mean a benign liver tumor characterized by well circumscribed nodules that consist of sheets of hepatocytes with a bubbly vacuolated cytoplasm. The hepatocytes are on a regular reticulin scaffold and less or equal to three cell thick. It is generally diagnosed by histological analysis. Subgroups of HCA 40 include "HNF1A" mutated HCA", which is a HCA characterized by the presence of mutation(s) in the HNF1A gene, "P catenin mutated HCA", which is a HCA WO 2014/044853 PCT/EP2013/069751 characterized by the presence of mutation(s) in the P catenin gene, "inflammatory HCA", which is a HCA characterized by presence of inflammatory infiltrate, sinusoidal dilatation, dystrophic arteries and overexpression of SAA protein at histological and immunohistochemical analysis, and "other HCA", which corresponds to a HCA sample 5 that is neither a HNF1A" mutated HCA, a P catenin mutated HCA, nor an inflammatory HCA.Other benign hepatocellular samples include healthy liver samples, cirrhotic liver samples, and regenerative macronodule samples (with or without dysplasia). By "regenerative macronodule", it is intended to mean liver nodules of more than 3 mm, which form in response to necrosis, altered circulation, or other stimuli, characterized 10 by benign hepatocyte with or without cell dysplasia. It is generally diagnosed by histological analysis. In the methods according to the invention, liver samples are analyzed. Such liver samples may notably be a liver biopsy or a partial or whole liver tumor surgical 15 resection. Reference samples used for calibrating algorithms and distance function are also liver samples, preferably of the same type as those analyzed. The above methods according to the invention are based on the in vitro determination of particular expression profiles comprising or consisting of specific genes. 55 genes 20 are needed for performing the most complete classification (non-hepatocellular; HCC with further classification into one of subgroups G1 to G6; FNH; HCA with further classification into HNF1A mutated HCA, inflammatory HCA, P catenin mutated HCA or other HCA; and other benign liver sample). Information concerning those 55 genes is provided in Table 2 below: 25 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR Activation of ANGPT2; CHKA; ADM Adrenomedullin 11p15.4 adrenomedullin pathway, ENO1; G6PD; HN1; angiogenesis, NPEPPS; RAN; TAF9 vasodilatation AFP Alpha-fetoprotein 4q11-q13 Foetal liver gene, stem CYP3A7; GPC3; HAL cell marker Aldo-keto reductase family ANGPTL7; CAP2; AKR1B10 1, member B10 (aldose 7q33 Reduction of aliphatic GPC3; PIR; SPP1; 1 embe i0aoand aromatic aldehydes TKT; reductase) AKR1 C1.AKR1 C2 GLUL; HAL; LAMA3; MERTK; MIA3; MME; Fatty acid degradation, PHB; PIR; REG3A; AMACR Alpha-rethylacyl-CoA 5p13.2-q11.1 peroxisomal beta- SLC16A1; SLPI; oxidation TBX3; AKR1 C1.AKR1 C2; HNF4A ANGPT1 Angiopoletin 1 8q23.1 Vascular development GIMAP5; KLRB1; and angiogenesis RAMP3 WO 2014/044853 10 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR BIRC5; CCNB1; CDC20; DPP8; G6PD; GLA; HN1; ANGPT2 Angiopoietin 2 8p23 Vascular development KPNA2; NEK7; and angiogenesis NEU1; NPEPPS; NRAS; RAN; SAE1; TRIP13; CKS2; DLGAP5 ANGPTL7 Angiopoietin-like 7 1p 36 Vascular development AKR1B10; ESR1; and angiogenesis GPC3; SPP1; TKT BIRC5; CCNB1; CDC20; GLA; HN1; AURKA Aurora kinase A 20q13 Cell cycle regulation, HSPA4; KPNA2; chromosome segregation NRAS; SAE1; TRIP13; CKS2; RRM2; DLGAP5 CYP2C9; ESR1; FABP1; G6PD; C8A Complement component 8, 1 p 3 2
.
2 Component of the GNMT; LCAT; alpha polypeptide complement system RARRES2; SAE1; UGT2B7; STEAP3; SERPIN CAP, adenylate cyclase- Interaction with adenylyl DPP8; HSPA4; MIA3; CAP2 associated protein, 2 6p22.3 cyclase-associated NEK7; NEU1; SAE1; (yeast) protein and actin TAF9 CCL5 Chemokine (C-C motif) 17q 11.2-q12 Immunoregulatory and G6PD; GIMAP5; ligand 5 inflammatory processes KLRB1; RAMP3 AURKA; BIRC5; CCNB1; G6PD; GLA; CDC20 Cell division cycle 20 1p34.1 Cell cycle regulation HN1; KPNA2; NRAS; homolog (S. cerevisiae) ' SAE1; TRIP13; CKS2; RRM2; DLGAP5 Cadherin 2, type 1, N- Calcium dependent cell MIA3; CDH2 cadherin 18q12.1 adhesion protein AKR1C1.AKR1C2; (neuronal) HNF1A FABP1; GNMT; Cytochrome P450, family 2 Drug metabolism and RHBG; UGT2B7; CYP2C9 , subfamily C, polypeptide 10q24.1 synthesis of cholesterol CKS2; C8A; 9 and steroids. AKR1C1.AKR1C2; SERPIN Cytochrome P450, family Drug and aflatoxin B1 AEP; CRP; CYP2C9; CYP3A7 3, 7q21-q22.1 metabolism, synthesis of GLS2; GPC3; HAL; subfamily A, polypeptide 7 cholesterol and steroids. SLPI AMACR; AURKA; BIRC5; CAP2; NADPH-dependent CCNB1; CHKA; DHRS2 Dehydrogenase/reductase 14qN1.2 dicarbonyl reductase GLUL; HAMP; (SDR family) member 2 arbony HSPA4; MIA3; PIR; (activity SLC16A1; TAF9; TBX3; RRM2; AKR1 C1.AKR1 C2 HN1; NPEPPS; NTS; EPCAM Epithelial cell adhesion 2p21 Membrane protein and RARRES2; TBX3; molecule liver stem cell marker C8A; KRT19; AKR1 C1.AKR1 C2 WO 2014/044853 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR Ephrin receptor subfamily YP3A7; GLS2; EPHA1 EPH receptor Al 7q32-q36 of the protein-tyrosine GLL STEAP3; kinase family RBM47 Estrogen binding, DNA AURKA; BIRC5; ESR1 Estrogen receptor 1 6q24-q27 binding, and activation of TRIB1 CKS2; transcription RRM2 CRP; CYP2C9; Fatty acid binding protein Binds free fatty acids and CYP3A7; GNMT; FABP1 2p11 their coenzyme A HAL; LCAT; UGT2B7; 1, derivatives C8A; HNF4A; SERPIN GOS2 GO/G1 switch 2 1q32.2-q41 Pro-apoptotic factor CXCR7; LGR5 GTPase, IMAP family GTP-binding superfamily, ANGPT1; CCL5; GIMAP5 member 5 7q36.1 mitochondrial integrity KLRB1; RAMP3; Regulation of glutamine CAP2; EPHA1; GLUL; GLS2 Glutaminase 2 (liver, 12q13 catabolism, mitochondrial GNMT; HAL; LAMA3; mitochondrial) respiration SDS; SLPI; STEAP3; CYP2C1 9 AMACR; CAP2; GLUL Glutamate-ammonia ligase 1q31 Synthesis of glutamine GLS2; GLUL HAL; REG3A; SLPI; TBX3 ARFGEF2; AURKA; BIRC5; CCNB1; CDC20; DPP8; Geminin DNA replication Inhibition of DNA G6PD; GLA; HN1; GMNN 'in 6p21.32 replication, cell cycle HSPA4; KPNA2; inhibitor regulation NEK7; NEU1; NRAS; PSMD1; RAN; SAE1; TAF9; TRIP13; CKS2; RRM2; DLGAP5 CYP2C9; FABP1; GNMT metycinsferase 6 p 12 Metabolism of methionine LCAT 2;RS; UGT2B7; CKS2; C8A AFP; AMACR; CRP; CYP3A7; EPHA1; HAL Histidine ammonia-lyase 12q22-q24.1 Histidine catabolism GLS2; GLUL; LAMA3; REG3A; SDS; SLPI; TBX3 AURKA; BIRC5; CAP2; CCNB1; CDC20; CRP; ESR1; Hepcidin antimicrobial Iron homeostasis, HSPA4; LCAT; NEK7; HAMP peptide 19q131 inflammation target gene SAA2; SAE1; TFRC; TRIP13; CKS2; STEAP3; RRM2; DLGAP5; LYVE1 WO 2014/044853 12 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR ARFGEF2; AURKA; BIRC5; C14orf156; CCNB1; CDC20; DNA replication, GLA; HN1; HSPA4; HMGB3 High mobility group box 3 Xq28 nucleosome assembly NA; RAN; SA; and transcription NRS TA9; TFRC; SLPI; TAF9; TFRC; TRIP13; CKS2; RRM2; DLGAP5; RBM47 AURKA; BIRC5; CCNB1; CDC20; Hematological and ENO1; G6PD; GLA; HN1 neurological 17q25.1 Regulation of androgen HSPA4; KPNA2; expressed 1 receptor NRAS; PDCD2; RAN; SAE1; TRIP13; CKS2; RRM2; DLGAP5 AMACR; CYP2C9; hepatocyte nuclear factor Transcription factor, liver FABP1; UGT2B7; HNF4A 4, alpha 20q13.12 development C8A; AKR1 C1.AKR1 C2; HNF1A; SERPIN BIRC5; CCNB1; Insulin-like growth factor 2 Translation repression of CDC20; G6PD; HN1; IGF2BP3 mRNA binding protein 3 7 p 1 5.3 insulin-like growth factor KPNA2; NRAS; n-lie grwthfactr 2SAEi; TRIPi 3; CKS2; RRM2 CYP2C9; GNMT; Structural integrity of HN1; IGF2BP3; KRT19 Keratin 19 17q21-q23 epithelial cells, liver stem NPEPPS;NTS; cell arker RARRES2; TBX3; cell marker C8A; EPCAM; AKR1 C1.AKR1 C2 AMACR; BIRC5; CAP2; CCNB1; CDC20; CHKA; DPP8; G6PD; GLA; LAMA3 Laminin, alpha 3 18q1.2 Cell adhesion and GLS2; GLUL; HAL; migration HN1; HSPA4; LGR5; NEK7; NPEPPS; NRAS; PSMD1; REG3A; SAE1; TAF9; TBX3; TKT; TRIP13 AURKA; BIRC5; Lysosomal protein Regulation of apoptosis CCNB1; CDC20; LAPTM4B transmembrane 8q22.1 and lysosomal KG6PD; HN ; SA4; 4 beta degradation SPA; TRIP1;RN SAE1; TRIP13; CKS2; DLGAP5 BIRC5; CCNB1; CDC20; ESR1; Lecithin-cholesterol Extracellular metabolism G6PD; GLA; GNMT; LCAT acyltransferase 16q22.1 of plasma lipoproteins HN1; NPEPPS; SPP1; TRIP13; CKS2; RRM2; C8A WO 2014/044853 13 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR AMACR; ANGPTL7; Leucine-rich repeat CHKA; GOS2; GLS2; LGR5 containing 12q22-q23 Wnt/catenin signaling GLUL; HAL; LAMA3; G protein-coupled MERTK; REG3A; receptor 5 RHBG; SDS; SLPI; TBX3 Lymphatic vessel AURKA; BIRC5; LYVE L endothelial Autocrine regulation of CCNB1; CDC20; hyaluronan receptor 1 cell growth, metastasis ESRI; HAMP; SAA2; TRIP13; RRM2 AMACR; CAP2; CRP; C-mer proto-oncogene Member of the GLS2; GLUL; HAL; MERTK ktyrosintoonase 2q14.1 MER/AXL/TYRO3 LAMA3; LGR5; MME; receptor kinase family NRAS; PSMD1; SLC16A1; TAF9 ARFGEF2; AURKA; BIRC5; CCNB1; CDC20; DPP8; Neuroblastoma RAS viral Oncogene, activation of ENO1; G6PD; GLA; NRAS (v-ras) 1p13.2 MAP kinase pathway HN1; HSPA4; oncogene homolog KIAA0090; KPNA2; PDCD2; PSMD1; RAN; SAE1; TAF9; TRIP13 NRCAM Neuronal cell adhesion 7q31 Cell adhesion molecule, CRP; G6PD; GNMT; molecule cell migration HN1; IGF2BP3; SPP1 AGPS; ARFGEF2; AURKA; BIRC5; C14orfl156; CCNB1; p21 protein (Cdc42/Rac)- Control of cell survival DPP8; ENO1; G6PD; PAK2 activated 3q29 and growth. Modulation of GLA HN1; NHSPA4; kinase 2 apoptosis. NEU; NRAS; PDCD2; PSMD1; RAN; SAE1; TAF9; TKT AKR1 B10; AMACR; AURKA; C14orf156; CAP2; CCNB1; Transcriptional ENO1; GLA; GLUL; PIR Pirin (iron-binding nuclear Xp22.31 coregulator, involve in HSPA4; KPNA2; protein) apoptosis and cell MIA3; NUDT9; migration PSMD1; RRAGD; SLC16A1; TAF9; TBX3; TKT; AKR1 C1.AKR1 C2 AGPS; ARFGEF2; C14orfl156; DPP8; ENO1; G6PD; GLA; Ras superfamily of HN1; HSPA4; RAB1A RAB1A, member RAS 2p14 GTPases, transit of KIAA0090; KPNA2; oncogene family protein through Golgi NEK7; NEU1; NRAS; compartment NUDT9; PAK2; PDCD2; PSMD1; RAN; SAE1; TAF9;
TFRC
WO 2014/044853 14 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR ANGPT1; BIRC5; CCL5; CCNB1; CYP2C9; ESR1; Receptor (G protein- Adrenomedullin receptor, GIMAP5; GNMT; RAMP3 coupled) 7p13-p12 vasodilatation, HAMP; KLRB1; activity modifying protein 3 angiogenesis LCAT; SDS; UGT2B7; CKS2; STEAP3; RRM2; CYP2C19; C8A C14orf156; CCNB1; Ras/raf pathway, control DPP8; ENO1; G6PD; RAN RAN, member RAS 12q24.3 of GLA; HN1; HSPA4; oncogene family DNA synthesis and cell KPNA2; NRAS; cycle progression PDCD2; PSMD1; SAE1; TAF9 CYP2C9; GNMT; Retinoic acid receptor LCAT; MIA3; RARRES2 responder (tazarotene 7q36.1 Chemotactic protein KRT19; EPA; AKR1 C1.AKR1 C2; SERPIN ARFGEF2; C14orf156; DPP8; RNA binding motif protein ENO1; HN1; HSPA4; RBM47 47 4p14 Unknown function KPNA2; NRAS; NUDT9; PDCD2; PSMD1; RAN; TAF9; TFRC Pancreatic secretory AMACR; EPHA1; REG3A Regenerating islet-derived 2p12 protein, involved in cell GLS2; GLUL; HAL; 3 alpha proliferation, also called LAMA3; LGR5; PAP RHBG; SLPI; TBX3 AMACR; CXCR7; CYP2C9; GLUL; HAL; RHBG Rh family, B glycoprotein 1q21.3 Ammonium transporter LAMA3; LGR5; (gene/pseudogene) REG3A; SLPI; TBX3; UGT2B7; AKR1 C1.AKR1 C2 AURKA; BIRC5; Protein of the acute CNB1; CRP; ESR1; SAA2 Serum amyloid A2 11p15.1-p14 phase of inflammation HAMP; NEK7; STEAP3; C8A; LYVE1 ARFGEF2; AURKA; BIRC5; CCNB1; CDC20; DPP8; SUMO1 activating enzyme Posttranslational G6PD; GLA; HN1; SAE1 subunit 1 1 9q13.32 modification of proteins, HSPA4; KPNA2; sumoylation NEK7; NRAS; PSMD1; RAN; TAF9; TRIP13; CKS2; RRM2; DLGAP5 CHKA; CRP; Metabolism of serine GADD45B; GLS2; SDS Serine dehydratase 12q24.21 glycine and ammoni' GLUL; GNMT; HAL; LGR5; RAMP3; SAA2; SLPI; C8A WO 2014/044853 15 PCT/EP2013/069751 Equivalent genes Gene short HUGO Gene name Chromosome Biological functions among the 103 genes name location tested in quantitative PCR TAF9 RNA polymerase ,transcriptional activation, ARFGEF2; CCNB1; TAF9 TATA box binding protein 5q11.2-q13.1 tr n ron nation DPP8; HSPA4; (TBP)-associated associated with apoptosis KPNA2; NRAS; RAN; factor, 32kDa SAE1 AURKA; BIRC5; CCNB1; CDC20; ENO1; G6PD; HN1; TFRC Transferrin r1eptor (p90' 3q26.2-qter Cellular uptake of iron HSPA4; KPNA2; NRAS; RAN; SAE1; TRIP13; CKS2; RRM2 UDP CRP; CYP2C9; UGT2B7 glucuronosyltransferase 2 4q13 Regulation of estrogen FABP1; GNMT; family polypeptide B7 metabolites RARRES2; C8A; fI , p d AKR1 C1.AKR1 C2 Table 2. Description of the 55 genes included in the classification algorithm, as well as genes considered as equivalents, i.e. the at most 10 genes which expression in HCC samples is best correlated to the original gene, with a Pearson's correlation coefficient > 0.3 or5 -0.3. 5 In the above methods according to the invention, in order to distinguish hepatocellular/non-hepatocellular samples, benign/malignant hepatocellular samples, FNH/non-FNH benign hepatocellular samples, HCA/non-HCA benign hepatocellular samples, HNF1A mutated/ non-HNF1A mutated HCA samples, inflammatory/non 10 inflammatory HCA samples, and P catenin mutated/non-p catenin mutated HCA samples, expression profiles comprising or consisting of specific genes, or Equivalent Expression Profiles thereof are analyzed. By "expression profile", it is meant the expression levels of the group of genes included in the expression profile. By "comprising", it is intended to mean that the expression profile may further comprise 15 other genes. In contrast, by "consisting of", it is intended to mean that no further gene is present in the expression profile analyzed. By "Equivalent Expression Profile thereof" or "EEP", it is intended to mean the original expression profile (to which said EEP is equivalent), wherein the addition, deletion or substitution of some of the genes (preferably at most 1 or 2 genes) does not change significantly the reliability of the 20 diagnosis, i.e. for which the values of sensitivity (Sen), specificity (Spe), positive predictive value (PPV), and negative predictive value (NPV) are not lowered by more than 10%. Sensitivity, specificity, PPV and NPV are usual statistical parameters well-known to those skilled in the art. 25 Sensitivity relates to the test's ability to identify positive results and is the proportion of people who have the disease who test positive for it. Specificity relates to the ability of the test to identify negative results and is defined as the proportion of patients who do not have the disease who will test negative for it.
WO 2014/044853 16 PCT/EP2013/069751 Positive predictive value (PPV) is the proportion of positive test results that are true positives. Negative predictive value (NPV) is defined as the proportion of subjects with a negative test result who are correctly diagnosed. 5 In a preferred embodiment, Equivalent Expression Profiles include expression profiles in which one of the genes of a selected genes combination is replaced by an equivalent gene. In the present description, a first gene ("gene A") can be considered as equivalent to another second gene ("gene B"), when replacing "gene A" in the expression profile of by "gene B" does not significantly impact the performance of the 10 test, i.e. the values of sensitivity (Sen), specificity (Spe), positive predictive value (PPV), and negative predictive value (NPV) are not lowered by more than 10%. This is typically the case when "gene A" is correlated to "gene B", meaning that the expression of "gene A" is statistically correlated to the expression level of "gene B", as determined by a measure such as Pearson's correlation coefficient. The correlation may be 15 positive (meaning that when "gene A" is upregulated in a patient, then "gene" B is also upregulated in that same patient) or negative (meaning that when "gene A" is upregulated in a patient, then "gene B" is downregulated in that same patient). A maximum of 10 genes among the 103 genes analyzed by the inventors using quantitative PCR, which are the best correlated to each of the 55 genes necessary for 20 complete classification, and which have an average Pearson's correlation coefficient> 0.3 or 5 -0.3 are mentioned in Table 2 above. By "determining an expression profile", it is meant the measure of the expression level of a group a selected genes. The expression level of each gene may be determined in 25 vitro either at the proteic or at the nucleic level, using any technology known in the art. For instance, at the proteic level, the in vitro measure of the expression level of a particular protein may be performed by any dosage method known by a person skilled in the art, including but not limited to ELISA or mass spectrometry analysis. These technologies are easily adapted to any liver sample. Indeed, proteins of the liver 30 sample may be extracted using various technologies well known to those skilled in the art for ELISA or mass spectrometry in solution measure. Alternatively, the expression level of a protein in a liver sample may be analyzed using mass spectrometry directly on the tissue slice. In a preferred embodiment of a method according to the invention, the expression 35 profile is determined in vitro at the nucleic level. At the nucleic level, the in vitro measure of the expression level of a gene may be carried out either directly on messenger RNA (mRNA), or on retrotranscribed complementary DNA (cDNA). Any method to measure the expression level may be used, including but not limited to microarray analysis, quantitative PCR, southern analysis. In a preferred embodiment of 40 a method according to the invention the expression profile is determined in vitro using a nucleic acid microarray, in particular an oligonucleotide microarray. In another WO 2014/044853 17 PCT/EP2013/069751 preferred embodiment of a method according to the invention, the expression profile is determined in vitro using quantitative PCR. In any case, the expression level of any gene is preferably normalized. There are many methods for normalizing obtained expression data, depending on the technology used for measuring expression. Such 5 methods are well known to those skilled in the art. In some embodiments, normalization may be performed in comparison to the expression level of an internal control gene, generally a household gene, including but not limited to ribosomal RNA (such as for instance 18S ribosomal RNA) or genes such as HPRT1 (hypoxanthine phosphoribosyltransferase 1), UBC (ubiquitin C), YWHAZ (tyrosine 3 10 monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), B2M (beta-2-microglobulin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), FPGS (folylpolyglutamate synthase), DECR1 (2,4-dienoyl CoA reductase 1, mitochondrial), PPIB (peptidylprolyl isomerase B (cyclophilin B)), ACTB (actin P), PSMB2 (proteasome (prosome, macropain) subunit, beta type, 2), GPS1 (G protein 15 pathway suppressor 1), CANX (calnexin), NACA (nascent polypeptide-associated complex alpha subunit), TAX1BP1 (Taxi (human T-cell leukemia virus type 1) binding protein 1), and PSMD2 (proteasome (prosome, macropain) 26S subunit, non-ATPase, 2). In the context of the present invention, "expression values" (also referred to as 20 "expression levels") of genes used for the prognosis include both: * non-normalized raw expression values, and * derivatives of raw expression values, which may further have been normalized no matter with method is used for normalization. In particular, when quantitative PCR is used for measuring in vitro expression 25 values of genes used for prognosis, derivatives of raw expression values selected from ACt, -ACt, AACt, or -AACt values may be used. When a microarray is used for measuring in vitro expression values of genes used for prognosis, log derivatives (in particular log2 derivatives) of raw expression values (which may furher have been normalized or not) are usually 30 used. These technologies are also easily adapted to any liver sample. Indeed, several well known technologies are available to those skilled in the art for extracting mRNA from a tissue sample and retrotranscribing mRNA into cDNA. 35 Many algorithms may be used for interpreting expression profiles in order to distinguish hepatocellular/non-hepatocellular samples, benign/malignant hepatocellular samples, FNH/non-FNH benign hepatocellular samples, HCA/non-HCA benign hepatocellular samples, HNF1A mutated/ non-HNF1A mutated HCA samples, inflammatory/non inflammatory HCA samples, and P catenin mutated/non-p catenin mutated HCA 40 samples. Notably, appropriate algorithms include PLS (Partial Least Square) regression, Support Vector Machines (SVM), linear regression or derivatives thereof WO 2014/044853 18 PCT/EP2013/069751 (such as the generalized linear model abbreviated as GLM, including logistic regression), Linear Discriminant Analysis (LDA, including Diagonal Linear Discriminant Analysis (DLDA)), Diagonal quadratic discriminant analysis (DQDA), Random Forests, k-NN (Nearest Neighbour) or PAM (Predictive Analysis of Microarrays) algorithms. 5 A group of reference samples, which is generally referred to as training data, is used to select an optimal statistical algorithm that best separates good from bad prognosis (like a decision rule). The best separation is usually the one that misclassifies as few samples as possible and that has the best chance to perform comparably well on a different dataset. 10 For a binary outcome such as good/bad prognosis, linear regression or a generalized linear model (abbreviated as GLM), including logistic regression, may be used. Linear regression is based on the determination of a linear regression function, which general formula may be represented as: f(xl,...,XN)= 0
+
1 1
X
1 +.+NXN" 15 Logistic regression is based on the determination of a logistic regression function: ef 1 e +1 1+ez in which z is usually defined as z=Bo +l x+...+BNxN In the above linear or logistic regression functions, x 1 to XN are the expression values 20 (or derivatives thereof such as ACt, -ACt, AACt, or -AACt for quantitative PCR or logged values for microarray) of the N genes in the signature, Po is the intercept, and P1 to PN are the regression coefficients. The values of the intercept and of the regression coefficients are determined based on a group of reference samples ("training data"). The value of the linear or logistic 25 regression function then defines the probability that a test expression profile has a good or bad prognosis (when defining the linear or logistic regression function based on training data, the user decides if the probability is a probability of good or bad prognosis). A test expression profile is then classified as having a good or bad prognosis depending if the probability that it has good or bad prognosis is inferior or 30 superior to a particular threshold value, which is also determined based on training data. Sometimes, two threshold values are used, defining an undetermined area. Other types of generalized linear models than logistic regression may also be used. Alternative methods such as nearest neighbour (abbreviated as k-NN) are also commonly used for a new sample, based on whether the sample is closer to the group 35 of good prognosis or to the group of bad prognosis. The notion of "closer" is based on a choice of distance (metric, such as but not limited to Euclidian distance) in the n dimension space defined by a signature consisting of N genes useful for prognosis (thus excluding potential housekeeping genes used for normalization purpose). The distances between a test expression profile and all reference good or bad prognosis WO 2014/044853 1 PCT/EP2013/069751 expression profiles are calculated and the sample is classified by analysis of the k closest reference samples (k being an positive integer of at least 1 and most commonly 3 or 5), a rule of classification being pre-established depending of the number of good or bad prognosis reference expression profiles among the k closest reference 5 expression profiles. For instance, when k is 1, a test expression profile is classified as good prognosis if the closest reference expression profile is a good prognosis expression profile, and as bad prognosis if the closest reference expression profile is a bad prognosis expression profile. When k is 2, a test expression profile is classified as responding if the two closest reference expression profiles are good prognosis 10 expression profiles, as non-responding if the two closest reference expression profiles are bad prognosis expression profiles, and undetermined if the two closest reference expression profiles include a good prognosis and a bad prognosis reference expression profile. When k is 3, a test expression profile is classified as good prognosis if at least two of the three closest reference expression profiles are good prognosis 15 expression profiles, and as bad prognosis if at least two of the three closest reference expression profiles are bad prognosis expression profiles. More generally, when k is p, a test expression profile is classified as good prognosis if more than half of the p closest reference expression profiles are good prognosis expression profiles, and as bad prognosis if more than half of the p closest reference expression profiles are bad 20 prognosis expression profiles. If the numbers of good prognosis and bad prognosis reference expression profiles are equal, then the test expression profile is classified as undetermined. Other methodologies from the field of statistics, mathematics or engineering exist, for example but not limited to decision trees, Support Vector Machines (SVM), Neural 25 Networks and Linear Discriminant Analyses (LDA). These approaches are well known to people skilled in the art. In summary, an algorithm (which may be selected from linear regression or derivatives thereof such as generalized linear models (GLM, including logistic regression), nearest neighbour (k-NN), decision trees, support vector machines (SVM), neural networks, 30 linear discriminant analyses (LDA), Random forests, or Predictive Analysis of Microarrays (PAM) is calibrated based on a group of reference samples (preferably including several good prognosis reference expression profiles and several bad prognosis reference expression profiles) and then applied to the test sample. In simple terms, a patient will be classified as good prognosis (or bad prognosis) based on how 35 all the genes in the signature compare to all the genes from a reference profile that was developed from a group of good prognosis (training data). The notion of whether individual genes of the expression profile are increased or decreased in a good prognosis versus a bad prognosis sample is of scientific interest. For each individual gene, the gene expression levels in the good prognosis group can 40 be compared to the bad prognosis group by the use of Student's t-test or equivalent WO 2014/044853 20 PCT/EP2013/069751 methods. However, such binary comparisons are generally not used for prognosis when a signature comprises several distinct genes. In a preferred embodiment, algorithm(s) used for interpreting any expression profile 5 described herein as useful for distinguishing the above mentioned samples are selected from: a) Prediction Analysis of Microarrays (PAM): PAM(sample X) = Arg max (yes (sample X); ONo (sample X)) wherein 6 Yes (sample X) = (1 'T X 7TYesji - Kyes i=1 6 No (sample X) = (1 - x N )Noi KNo i=1 wherein: 10 e xi, 1 i N, represent the in vitro measured values of N variables derived from the expression levels of genes of the expression profile, and e 7T, yi, 7Tyes,i, 7TNo,, 1 i N, Kyes and KNo are fixed parameters calibrated with at least one reference sample; 15 b) Diagonal Linear Discriminant Analysis (DLDA): DLDA(sample X) = Arg min(Ayes (sample X); ANo (sample X)) wherein Ayes (sample X) = (xi ANo (sample X) = (Xi - INo,i)2 1 1 wherein: * xi, 1 i N, represent the in vitro measured values of N variables derived from the expression levels of genes of the expression 20 profile, and e Ui, IpYes,i, and IINo,i, 1 i N, are fixed parameters calibrated with at least one reference sample; c) Diagonal quadratic discriminant analysis (DQDA): DQDA(sample X) = Arg min (VYes (sample X); VNo ( sample X)) wherein WO 2014/044853 21 PCT/EP2013/069751 (si (N
-
' e
)
2 Vyes(sample X) = Ye i + CYes S VYesJi VNo(sample X) = (X [NNoi ) + CNo wherein: 0 xi, 1 i N, represent the in vitro measured values of N variables derived from the expression levels of genes of the expression profile, and 5 e uYesi, uNo,i, IYes,i, INo,i, , 1 i N, are fixed parameters calibrated with at least one reference sample, and C =es = log(VYes,) CNo =1 (ogVNo,i)); d) or any combination thereof. 10 For the purpose of interpreting expression profiles in order to distinguish hepatocellular/non-hepatocellular samples, benign/malignant hepatocellular samples, FNH/non-FNH benign hepatocellular samples, HCA/non-HCA benign hepatocellular samples, HNF1A mutated/ non-HNF1A mutated HCA samples, inflammatory/non inflammatory HCA samples, and P catenin mutated/non-p catenin mutated HCA 15 samples, a particularly advantageous algorithm is: Diagnosis (sample X) = majority rule (PAM(sample X), DLDA(sample X), DQDA(sample X)) In a preferred embodiment, for the purpose of interpreting expression profiles in order to distinguish hepatocellular/non-hepatocellular samples, benign/malignant hepatocellular samples, FNH/non-FNH benign hepatocellular samples, HCA/non-HCA 20 benign hepatocellular samples, HNF1A mutated/ non-HNF1A mutated HCA samples, inflammatory/non-inflammatory HCA samples, and P catenin mutated/non-p catenin mutated HCA samples, the expression profile(s) is(are) determined using quantitative PCR and the variables and parameters of PAM, DLDA and DQDA algorithms are the following: 25 a) For determining if a liver sample is or not a hepatocellular sample: e 6 variables x, to x6 are used as follows: xi (-AACt TFRC expression level) - (-ACt C8A expression level) xz (-AACt AFP expression level) + (-AACt GNMT expression level) x3 (-AACt HAL expression level) - (-ACt EPCAM expression level) x4 (-AACt CYP3A7 expression level) - (-AACt EPCAM expression level) WO 2014/044853 22 PCT/EP2013/069751 x5 (-MACt FABP1 expression level) - (-MACt EPCAM expression level) x6 (-MACt EPCAM expression level) - (-MACt HNF4A expression level) e PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi 1.342931 -0.09325912 2.006058 7.153821 xz -1.551583 0.10774885 -4.1733248 9.685958 x3 -1.23594 0.08582914 -0.9310016 10.17258 8.151418 0.0932632 x4 -1.524252 0.10585085 2.8897574 10.391148 x5 -1.261254 0.08758709 -1.0531553 10.049158 X6 1.087001 -0.07548619 -1.4702021 9.901341 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi VNo,i VYesi Vi Xi 11.613149 1.3388989 11.690171 4.251989 4.692407 xz -19.201897 -3.12967394 12.73627 22.662048 22.074337 x3 -13.503695 -0.05789783 17.965523 27.445047 26.883759 x4 -12.948974 3.98966931 6.765985 30.609874 29.198065 X5 -13.727697 -0.17297876 17.267584 26.144739 25.619118 X6 9.292567 -2.21761661 1.913791 25.543753 24.14461 b) For determining if a hepatocellular sample is or not a HCC sample: * 6 variables x, to x6 are used as follows: xi (-ACt CAP2 expression level) - (-ACt LCAT expression level) xz (-ACt ANGPT2 expression level) + (-AACt AURKA expression level) x3 (-ACt CDC20 expression level) + (-MACt DHRS2 expression level) x4 (-ACt ANGPT2 expression level) - (-ACt LYVE1 expression level) x5 (-ACt ADM expression level) - (-ACt CDC20 expression level) x6 Max (-ACt AFP expression level; -ACt CAP2 expression level) 5 e PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes x, -0.16268042 0.08134021 5.787048 4.542418 xz -0.22453753 0.11226876 3.035909 3.975872 x3 -0.42378458 0.21189229 3.937962 6.248688 1.272916 0.449041 x4 -0.2592874 0.1296437 4.151425 3.70769 X5 0.15685585 -0.07842792 -4.403932 3.840179 X6 -0.01726311 0.00863156 3.696066 4.123495 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi UNo,i UYesi Vi x1 2.678847 7.341149 2.2201 8.37556 6.33819 WO 2014/044853 23 PCT/EP2013/069751 Xz 0.06943705 4.519144 3.255149 4.0793 3.806517 x3 -1.96933307 6.891609 25.818236 13.894186 17.840878 X4 1.25620635 5.599034 1.863177 3.311281 2.831979 X5 -1.79861246 -5.706591 2.246134 3.814584 3.295449 X6 1.47414444 4.807026 1.020023 6.078697 4.404347 c) For determining if a benign hepatocellular sample is or not a FNH sample: * 12 variables x1 to Xi2 are used as follows: x, Min (-AACt ANGPTL7 expression level; -ACt GLUL expression level) xz (-AACt ANGPT1 expression level) - (-AACt HMGB3 expression level) x3 (-AACt GMNN expression level) + (-AACt RAMP3 expression level) x4 Min (-AACt RHBG expression level; -AACt UGT2B7 expression level) x5 Max (-AACt HAL expression level; -AACt RAMP3 expression level) x6 Min (-AACt LGR5 expression level; -AACt UGT2B7 expression level) x7 (-AACt RAMP3 expression level) + (-ACt UGT2B7 expression level) x8 (-AACt RAMP3 expression level) + (-ACt RARRES2 expression level) x9 Max (-AACt ANGPT1 expression level; -AACt RAMP3 expression level) xo Min (-AACt ANGPT1 expression level; -AACt LGR5 expression level) Xi1 (-ACt RAMP3 expression level) - (-ACt RBM47 expression level) Xi2 Min (-MACt GIMAP5 expression level; -ACt UGT2B7 expression level) e PAM parameters are the following: Xi 7TNo,i TYes,i i Yi KNo KYes x, -0.18469273 1.0817717 -1.72829395 3.243668 xz -0.15724871 0.9210281 0.61243528 2.336453 x3 -0.13637923 0.7987926 1.58326744 2.289755 x4 -0.15358836 0.899589 -3.46104209 3.909901 X5 -0.11234999 0.65805 1.19490255 2.017152 X6 -0.11945816 0.6996835 -2.27683325 3.334501 0.2800792 6.1260851 X7 -0.15338781 0.8984143 -0.04692744 2.922347 X8 -0.14256206 0.8350063 0.60258802 2.277919 X9 -0.11634108 0.6814263 1.54744785 1.913217 x10 -0.17351058 1.0162762 -1.4122167 3.581967 xii -0.15477031 0.9065118 1.45598643 2.048925 X12 -0.07438928 0.4357086 -1.04952428 2.524675 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi UNo,i UYesi Vi xi -2.3273759 1.7806145 4.6402628 0.60826433 4.11435 Xz 0.245031 2.76437457 1.4145492 0.20686229 1.2570248 X3 1.2709924 3.41230679 1.2978397 0.19883833 1.1544917 WO 2014/044853 24 PCT/EP2013/069751 X4 -4.0615574 0.05626186 8.3471726 0.0196296 7.2609714 X5 0.9682756 2.52228907 0.6935121 0.30621156 0.6429946 X6 -2.6751666 0.05626186 5.1618051 0.0196296 4.4910865 X7 -0.4951798 2.57855093 3.3012094 0.33314121 2.9140701 X8 0.2778432 2.50466495 1.2384457 0.40087507 1.1291973 X9 1.3248621 2.85116431 0.5424233 0.11837803 0.487113 x10 -2.0337258 2.22805082 6.3954525 0.30614496 5.601195 x11 1.1388737 3.31336105 0.7211325 0.52047864 0.6949603 X12 -1.2373331 0.05049854 1.9692555 0.01620956 1.7145104 d) For determining if a benign hepatocellular sample is or not a HCA sample: * 10 variables x, to xio are used as follows: xi (-MACt AKR1B10 expression level) + (-MACt GLS2 expression level) xz (-ACt LCAT expression level) - (-ACt KRT19 expression level) x3 (-ACt ESR1 expression level) + (-ACt SDS expression level) x4 Max (-ACt MERTK expression level; -ACt LYVE1 expression level) x5 Max (-ACt EPHAl expression level; -ACt KRT19 expression level) x6 (-ACt CCL5 expression level) + (-MACt GLS2 expression level) x7 (-ACt HAL expression level) - (-MACt MERTK expression level) x8 (-ACt CYP2C9 expression level) - (-MACt MERTK expression level) x9 (-ACt CCL5 expression level) + (-MACt KRT19 expression level) xo Min (-ACt CYP3A7 expression level; -ACt EPHAl expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi 1.1300586 -0.52467006 -0.96573089 5.405409 xz -0.6257754 0.29053858 0.10777331 4.174906 x3 -0.583684 0.27099612 1.53413349 3.92968 x4 -0.2101061 0.09754928 0.01545178 2.53848 X5 0.4031816 -0.18719147 0.76400666 2.906802 X6 0.6342941 -0.29449369 -1.82990856 4.756332 3.0655113 0.7945744 X7 0.5211003 -0.24193944 -0.57174662 4.026102 X8 0.3773559 -0.17520095 -0.97286634 3.529012 X9 0.8070427 -0.3746984 -0.75070901 3.946451 x10 0.3875215 -0.17992069 0.02720304 2.927056 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi UNo,i UYesi Vi X1 5.142698 -3.8017871 1.9223207 16.202619 11.8086811 X2 -2.5047803 1.3207446 4.8696186 4.8642148 4.8658775 X3 -0.759558 2.5990617 1.5948539 4.8438216 3.8441392 WO 2014/044853 25 PCT/EP2013/069751 X4 -0.5178985 0.2630787 0.1157701 0.4169368 0.3242701 X5 1.9359758 0.2198781 0.9741474 0.8373057 0.8794108 X6 1.1870048 -3.2306184 0.5402267 10.9818415 7.769037 X7 1.5262567 -1.5458196 1.0506355 5.6452689 4.2315355 X8 0.358827 -1.5911525 0.2637763 3.3978705 2.4335338 X9 2.4342454 -2.2294378 3.9252834 3.9034702 3.910182 X10 1.1615001 -0.4994349 0.507857 1.1000088 0.9178082 e) For determining if a HCA sample is or not a HNF1A mutated HCA sample: * 2 variables x, to x6 are used as follows: xi (-MACt DHRS2 expression level) - (-MACt UGT2B7 expression level) xz (-MACt ANGPT2 expression level) + (-MACt FABP1 expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes X1 -0.2597076 1.817954 -1.130125 6.501417 0.1803095 4.3715711 Xz -0.1615805 1.131063 1.136677 3.83618 5 e DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi UNo,i UYesi Vi X1 -2.8185929 10.68915 15.46252 14.3631833 15.343027 Xz 0.5168253 5.47564 1.668767 0.7321017 1.566956 f) For determining if a HCA sample is or not an inflammatory HCA sample: * 4 variables x, to x6 are used as follows: xi (-MACt HAMP expression level) + (-MACt SAA2 expression level) xz (-ACt CCL5 expression level) - (-ACt NRCAM expression level) x3 Max (-ACt EPHAl expression level; -ACt KRT19 expression level) x4 (-ACt ANGPT2 expression level) + (-ACt SAA2 expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi -0.4760712 0.9521423 4.6430007 6.107883 Xz 0.434627 -0.869254 -0.0574931 5.002872 0.7344381 2.4145044 x3 0.1882468 -0.3764937 1.1521703 3.158128 x4 -0.4549338 0.9098677 4.5882009 4.501345 10 e DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi UNo,i UYesi Vi x1 1.735214 10.4585747 16.9585649 7.6603747 13.9265464 WO 2014/044853 26 PCT/EP2013/069751 Xz 2.11689 -4.4062595 7.0569419 6.5761749 6.90017 X3 1.746678 -0.0368447 0.7298408 0.3673544 0.6116387 X4 2.540387 8.6838292 4.4787841 4.5955546 4.5168614 g) For determining if a HCA sample is or not a P catenin mutated HCA sample: * 9 variables x, to x6 are used as follows: x, (-MACt AKR1B1O expression level) - (-MACt REG3A expression level) x2 (-MACt AMACR expression level) + (-MACt HAL expression level) x3 (-ACt CAP2 expression level) - (-ACt GLUL expression level) x4 (-ACt HAL expression level) + (-ACt TAF9 expression level) xs (-ACt CAP2 expression level) - (-ACt LGR5 expression level) x6 Min (-ACt AKR1B1O expression level; -ACt HAL expression level) x7 (-ACt LAPTM4B expression level) + (-ACt TFRC expression level) x8 (-ACt GIMAP5 expression level) - (-ACt HAL expression level) x9 (-ACt HMGB3 expression level) - (-ACt IGF2BP3 expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi 0.34708654 -1.9668237 1.94438201 7.392962 xz 0.21863143 -1.2389115 -1.04516656 3.127947 x3 0.18579207 -1.0528217 1.22379671 2.663529 x4 0.24406366 -1.3830274 0.05214403 3.244264 Xs 0.15694722 -0.8893676 2.7521494 3.869139 0.3607787 8.2634614 X6 0.21470021 -1.2166345 -1.47714108 4.260375 X7 0.11140632 -0.6313025 0.81968112 3.203963 X8 -0.22080529 1.25123 0.49103172 3.193991 X9 0.04764503 -0.2699885 0.56180483 3.025541 5 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi UNo,i UYesi Vi Xi 4.5103796 -12.5962709 37.671414 6.2381109 33.535453 X2 -0.361299 -4.920416 1.426277 8.2837077 2.328571 X3 1.7186592 -1.5804241 1.203395 0.6218992 1.126882 X4 0.8439509 -4.4347616 1.358794 11.5298442 2.69709 Xs 3.3594 -0.6889375 5.646265 1.7986761 5.140003 X6 -0.5624378 -6.6604599 6.819184 8.7029888 7.067053 X7 1.1766229 -1.2029889 2.912529 0.2815287 2.566345 X8 -0.2142184 4.4874493 1.580383 8.8316336 2.534495 X9 0.7059568 -0.2550566 2.287403 0.3047094 2.026522 WO 2014/044853 27 PCT/EP2013/069751 The present invention also relates to a kit comprising reagents for the determination of an expression profile comprising at most 65 distinct genes, wherein said expression profile is selected from: " An expression profile comprising or consisting of the following 38 genes: 5 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control gene, or an 10 Equivalent Expression Profile thereof; * An expression profile comprising or consisting of the following 46 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, 15 RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control gene, or an Equivalent Expression Profile thereof; * An expression profile comprising or consisting of the following 49 genes: 20 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, 25 CDH2, HAMP, and SAE1, and optionally one or more internal control gene, or an Equivalent Expression Profile thereof; or * An expression profile comprising or consisting of the following 55 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, 30 ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, IGF2BP3, RAB1A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, and SAE1, and optionally one or more internal control gene, or an Equivalent Expression 35 Profile thereof. The kit according to the invention is preferably dedicated to the determination or one of the above mentioned expression profiles, and thus comprises reagents for the determination of an expression profile comprising at most 65 distinct genes, knowing 40 that the expression profile with the highest number of genes of interest comprises 55 genes, and optionally one or more internal control gene. When the expression profile WO 2014/044853 28 PCT/EP2013/069751 comprises less than 55 genes of interest, the kit preferably comprises reagents for the determination of an expression profile comprising the number of genes of interest and no more than about 10 additional genes, which may include internal control genes and/or a few additional genes. Such additional genes might correspond to a further 5 expression profile that might be used for instance for prognosis of the disease if the sample is determined as a HCC sample. For instance, when the expression profile comprises 49 genes of interest and optionally one or more internal control gene, the kit preferably comprises reagents for the determination of an expression profile comprising at most 59 distinct genes. When the 10 expression profile comprises 46 genes of interest and optionally one or more internal control gene, the kit preferably comprises reagents for the determination of an expression profile comprising at most 56 distinct genes. When the expression profile comprises 38 genes of interest and optionally one or more internal control gene, the kit preferably comprises reagents for the determination of an expression profile comprising 15 at most 48 distinct genes. In all the above mentioned embodiments of a kit comprising reagents for the determination of an expression profile comprising at most N distinct genes, N being an integer as mentioned above, reagents comprised in the kit do not permit determination of an expression profile comprising more than N genes. In particular, such a kit 20 according to the invention excludes pangenomic microarrays permitting determination of expression profiles of thousands of genes. Reagents for the determination of an expression profile comprising N genes may include any reagents permitting to specifically quantify the expression levels of the genes included in said expression profile. For instance, when the expression profile is 25 determined at the proteic level, then such reagents may include antibodies specific for each of the genes included in the expression profile. Preferably, the expression is determined at the nucleic level. In this case, reagents in the kit of the invention may notably include primers pairs (forward and reverse primers) and/or probes specific for each of the genes included in the expression profile (useful notably for quantitative 30 PCR determination of the expression profile) or a nucleic acid microarray, in particular an oligonucleotide microarray. In the latter case, the nucleic acid microarray is a dedicated nucleic acid microarray, comprising probes for the detection of a maximum number of genes, as defined in the previous paragraph. In other words, the nucleic acid microarray does not permit determination of an expression profile comprising more 35 than the maximum number of genes comprised in the expression profile. As indicated in introduction, the classification method according to the invention is important for clinicians because it will permit them, based on a unique and simple test, to know precisely of which type of liver disease a subject is suffering, and thus to adapt 40 the treatment to the precise diagnosis.
WO 2014/044853 29 PCT/EP2013/069751 The invention thus also relates to an IGFR1 inhibitor, an Akt/mTor inhibitor, a proteasome inhibitor and/or a wnt inhibitor, for use in the treatment of HCC in a subject that has been diagnosed as suffering from HCC based on a liver sample that has been classified as a HCC sample by the classification method of the invention. The invention 5 also relates to the use of an IGFR1 inhibitor, an Akt/mTor inhibitor, aproteasome inhibitor and/or a wnt inhibitor for the preparation of a medicament intended for the treatment of HCC in a subject that has been diagnosed as suffering from HCC based on a liver sample that has been classified as a HCC sample by the classification method of the invention. If the liver sample of said subject has been further classified 10 as subgroup G1, then a IGFR1 inhibitor or an Akt/mTor inhibitor is preferred. If the liver sample of said subject has been further classified as subgroup G2, then an Akt/mTor inhibitor is preferred. If the liver sample of said subject has been further classified as subgroup G3, then a proteasome inhibitor is preferred. If the liver sample of said subject has been further classified as subgroup G5 or G6, then a wnt inhibitor is 15 preferred. However, current WNT inhibitors have toxicity problems, and there is still a need for more efficient and safer WNT inhibitors. The invention also relates to a method for treating a liver disease in a subject in need thereof, comprising: 20 a) Classifying a liver sample of said subject as a non-hepatocellular sample, a hepatocellular carcinoma (HCC) sample, a focal nodule dysplasia (FNH) sample, a hepatocellular adenoma (HCA) sample or another benign liver sample with the classification method according to the invention; b) If said sample is a non-hepatocellular sample, then identifying the precise 25 histological subtype of sample and administering to said subject a treatment according to the histological subtype identified; c) If said sample is a HCC sample, then performing surgical resection with or without adjuvant treatment; d) If said sample is a FNH sample, then no therapeutic action is performed; 30 e) If said sample is a HCA sample, then only following up the subject or performing surgical resection, depending on the HCA subgroup; f) If said sample is another benign hepatocellular sample, then no therapeutic action is performed. The method of treatment of the invention may further comprise, if said liver sample is a 35 HCC sample: i. classifying said HCC sample into one of subgroups G1 to G6 as described above; and ii. if said HCC sample is classified in G1 subgroup, then administering an efficient amount of an IGFR1 inhibitor or of an Akt/mTor inhibitor to said patient; 40 iii. if said HCC sample is classified in G1-G2 subgroup, administering an efficient amount of an hen Akt/mTor inhibitor to said patient; WO 2014/044853 30 PCT/EP2013/069751 iv. if said HCC sample is classified in G3 subgroup, then administering an efficient amount of a proteasome inhibitor to said patient; v. if said HCC sample is classified in G5-G6 subgroup, then administering an efficient amount of a wnt inhibitor to said patient. 5 The method of treatment of the invention may further comprise, if said liver sample is a HCC sample: i. Prognosing global survival and/or survival without relapse; and ii. if said HCC sample is given a good prognosis, then no adjuvant treatment is performed; 10 iii. if said HCC sample is given a bad prognosis, then administering to said subject an adjuvant treatment, such as cytotoxic chemotherapy and/or targeted therapy. According to the invention, a "prognosis" of HCC evolution means a prediction of the future evolution of a particular HCC tumor relative to the patient suffering of this 15 particular HCC tumor. The method according to the invention allows simultaneously for both a global survival prognosis and a survival without relapse prognosis. By "global survival prognosis" is meant prognosis of survival, with or without relapse. As stated before, the main current treatment against HCC is tumor surgical resection. As a result, a "bad global survival prognosis" is defined as the occurrence of death 20 within the 3 years after liver resection, whereas a "good global survival prognosis" is defined as the lack of death during the 5 post-operative years. By "survival without relapse prognosis" is meant prognosis of survival in the absence of any relapse. A "bad survival without relapse prognosis" is defined as the presence of tumor-relapse within the two years after liver resection, whereas a "good survival 25 without relapse prognosis" is defined as the lack of relapse during the 4 post-operative years. Such prognosis of global survival and/or survival without relapse may be performed using any suitable method. Examples of such methods are notably described in W02007/063118A1. 30 Adjuvants treatments are administered in case of bad prognosis. Said adjuvant treatment may be selected from: a) cytotoxic chemotherapy, i.e. therapy with any suitable chemical agent useful for killing cancer cells. Cytotoxic chemotherapeutic agents currently used as adjuvant treatment of HCC and preferred in the present invention are 35 doxorubicin, gemcitabine, oxaliplatine, and combinations thereof. Doxorubicin or association of gemcitabine and oxaliplatine are particularly preferred. b) targeted therapy, i.e. therapy with any suitable agent that selectively inhibits enzymes of a signaling pathway involved in HCC malignant transformation. Currently, Sorafenib, a small molecular inhibitor of several Tyrosine protein 40 kinases (VEGFR and PDGFR) and Raf kinases (more avidly C-Raf than B- WO 2014/044853 31 PCT/EP2013/069751 Raf), is approved for the adjuvant treatment of HCC is is preferred in the present invention. Sorafenib is a bi-aryl urea of formula: CI HN F 0F N H 5 The method of treatment of the invention may also further comprise, if said liver sample is a HCA sample: i. classifying said HCA sample into one of subgroups HNF1A mutated HCA, inflammatory HCA, P catenin mutated HCA or other HCA as described above; 10 and ii. if said HCA sample is classified as a HNF1A mutated HCA sample, then only following up said subject if HCA < 5 cm, or performing surgical resection if HCA > 5 cm; iii. if said HCA sample is classified as an inflammatory HCA sample, then only 15 following up said subject if HCA < 5 cm, or performing surgical resection if HCA > 5 cm; iv. if said HCA sample is classified as a P catenin mutated HCA sample, then performing surgical resection whatever the HCA size. 20 The present invention also relates to systems (and computer readable medium for causing computer systems) to perform a method of classification of liver samples according to the invention. In an embodiment, the invention relates to a system 1 for classifying a liver sample comprising: 25 a) a determination module 2 configured to receive a liver sample and to determine expression level information concerning: " An expression profile comprising or consisting of the following 38 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, 30 GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; " An expression profile comprising or consisting of the following 46 genes: 35 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, WO 2014/044853 32 PCT/EP2013/069751 RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; 5 * An expression profile comprising or consisting of the following 49 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, 10 MERTK, EPHA1, CCL5, CYP2C9, RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and SAE1, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; or An expression profile comprising or consisting of the following 55 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, 15 LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, IGF2BP3, RAB1A, NRAS, PIR, LAMA3, GOS2, 20 HN1, PAK2, CDH2, and SAE1, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof. b) a storage device 3 configured to store the expression level information from the determination module; c) a comparison module 4, adapted to compare the expression level information 25 stored on the storage device with reference data, and to provide a comparison result, wherein the comparison result is indicative of the type of liver sample; and d) a display module 5 for displaying a content 6 based in part on the classification result for the user, wherein the content is a signal indicative of the type of liver 30 sample. In another embodiment, the invention relates to a computer readable medium 7 having computer readable instructions recorded thereon to define software modules for implementing on a computer steps of a classification method according to the invention relating to interpretation of expression profiles data. Preferably, said software modules 35 comprising: a) an entry module 8, which permits expression level information to be entered by a user and to be stored (at least temporarily) for further comparison, wherein said expression level information relates to: * An expression profile comprising or consisting of the following 38 genes: 40 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, WO 2014/044853 PCT/EP2013/069751 GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; 5 * An expression profile comprising or consisting of the following 46 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, 10 MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; An expression profile comprising or consisting of the following 49 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, 15 LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and SAE1, and optionally one or more 20 internal control genes, or an Equivalent Expression Profile thereof; or * An expression profile comprising or consisting of the following 55 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, 25 RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, IGF2BP3, RAB1A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, and SAE1, and optionally one or more internal control genes, or an Equivalent Expression Profile thereof; 30 b) a comparison module 4, adapted to compare the expression level information entered by the user with reference data and to provide a comparison result, wherein the comparison result is indicative of the type of liver sample; and c) a display module 5, for displaying a content 6 based in part on the comparison result for the user, wherein the content is a signal indicative of the type of liver 35 sample. Embodiments of the invention relating to systems and computer-readable media have been described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to 40 perform method steps when executed. The modules have been segregated by function for the sake of clarity. However, it should be understood that the modules need not WO 2014/044853 PCT/EP2013/069751 correspond to discreet blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times. Furthermore, it should be appreciated that the modules may perform other functions, thus the modules are not limited to having any particular functions or 5 set of functions. The computer readable medium can be any available tangible media that can be accessed by a computer. Computer readable medium includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data 10 structures, program modules or other data. Computer readable medium includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (eraseable programmable read only memory), EEPROM (electrically eraseable programmable read only memory), flash memory or other memory technology, CD ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical 15 storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing. Computer-readable data embodied on one or more computer-readable media, may 20 define instructions, for example, as part of one or more programs, that, as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein (e.g., in relation to system 1, or computer readable medium 7), and/or various embodiments, variations and combinations thereof. Such instructions may be written in any of a plurality of programming languages, for example, Java, J#, 25 Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof. The computer-readable media on which such instructions are embodied may reside on one or more of the components of either system 1, or computer readable medium 6 described herein, may be distributed across one or more of such components, and may be in transition there 30 between. The computer-readable media may be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein. In addition, it should be appreciated that the instructions stored on the computer readable media, or the computer-readable 35 medium, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the present invention. The computer executable instructions may be written in a suitable computer language or 40 combination of several languages. Basic computational biology methods are known to those of ordinary skill in the art and are described in, for example, Setubal and WO 2014/044853 35 PCT/EP2013/069751 Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997, ref 38); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998, ref 39); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC 5 Press, London, 2000, ref 40) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2 nd ed., 2001). The functional modules of certain embodiments of the invention include a determination module 2, a storage device 3, a comparison module 4 and a display module 5. The functional modules can be executed on one, or multiple, computers, or 10 by using one, or multiple, computer networks. The determination module 2 has computer executable instructions to provide expression level information in computer readable form. As used herein, "expression level information" refers to information about expression level of any nucleotide (RNA or DNA) and/or amino acid sequences, either full-length 15 or partial. In a preferred embodiment, it refers to the level of expression of mRNA or cDNA, measured by various technologies. The information may be qualitative (presence or absence of a transcript) or quantitative. Preferably it is quantitative. Methods for determining expression level information, i.e. determination modules 2, include systems for protein and DNA/RNA analysis, and in particular those described 20 above for determination of expression profiles at the nucleic or protein level. The expression level information determined in the determination module can be read by the storage device 3. As used herein the "storage device" 3 is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with 25 the present invention include stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and local and distributed computer processing systems. Storage devices 3 also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage 30 media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as magnetic/optical storage media. The storage device 3 is adapted or configured for having recorded thereon expression level information. Such information may be provided in digital form that can be transmitted and read electronically, e.g., via the 35 Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication including wireless communication between devices. As used herein, "stored" refers to a process for encoding information on the storage device 3. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the 40 expression level information.
WO 2014/044853 36 PCT/EP2013/069751 A variety of software programs and formats can be used to store the expression level information on the storage device. Any number of data processor structuring formats (e.g., text file, spreadsheets or database) can be employed to obtain or create a medium having recorded thereon the expression level information. 5 By providing expression level information in computer-readable form, one can use the expression level information in readable form in the comparison module 4 to compare a specific expression profile with the reference data within the storage device 3. The comparison may notably be done using the various algorithms described above. The comparison made in computer-readable form provides a computer readable 10 comparison result which can be processed by a variety of means. Content based on the comparison result can be retrieved from the comparison module 4 and displayed by the display module 5 to indicate the type of liver sample. Preferably, reference data are expression level profiles that are indicative of all types of liver samples that may be found by a classification method according to the invention. 15 The "comparison module" 4 can use a variety of available software programs and formats for the comparison operative to compare expression level information determined in the determination module 2 to reference data, either directly, or indirectly using any software providing statistical classification algorithms such as those already described above. 20 The comparison module 4, or any other module of the invention, may include an operating system (e.g., Windows, Linux, Mac OS or UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server. World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) 25 statements). Generally, the executables will include embedded SQL statements. In addition, the World Wide Web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The Configuration file also directs requests for server 30 resources to the appropriate hardware--as may be necessary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as "Intranets." An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., 35 the GenBank or Swiss Pro World Wide Web site). Thus, in a particular preferred embodiment of the present invention, users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers. The comparison module 4 provides computer readable comparison result that can be 40 processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a content 6 based in part on the comparison result that may be stored WO 2014/044853 37 PCT/EP2013/069751 and output as requested by a user using a display module 5. The display module 5 enables display of a content 6 based in part on the comparison result for the user, wherein the content is a signal indicative of the type of liver sample. Such signal can be, for example, a display of content indicative of the type of liver sample on a 5 computer monitor, a printed page or printed report of content indicating the type of liver sample from a printer, or a light or sound indicative of the type of liver sample. The display module 5 can be any suitable device configured to receive from a computer and display computer readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel 10 PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or from ARM Holdings, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types or integrated devices such as laptops 15 or tablets, in particular iPads. In one embodiment, a World Wide Web browser is used for providing a user interface for display of the content 6 based on the comparison result. It should be understood that other modules of the invention can be adapted to have a web browser interface. Through the Web browser, a user may construct requests for retrieving data from the 20 comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces. The requests so formulated with the user's Web browser are transmitted to a Web application which formats them to produce a query that can be employed to extract the pertinent information. 25 In one embodiment, the display module 5 displays the comparison result and whether the comparison result is indicative of the type of liver sample. In one embodiment, the content 6 based on the comparison result that is displayed is a signal (e.g. positive or negative signal) indicative of the type of liver sample, thus only a positive or negative indication may be displayed. 30 The present invention therefore provides for systems 1 (and computer readable media 7 for causing computer systems) to perform methods of classifying liver samples, based on expression profiles information. System 1, and computer readable medium 7, are merely illustrative embodiments of the invention for performing methods of classification of liver sample based on 35 expression profiles, and are not intended to limit the scope of the invention. Variations of system 1, and computer readable medium 7, are possible and are intended to fall within the scope of the invention. The modules of the system 1 or used in the computer readable medium, may assume numerous configurations. For example, function may be provided on a single machine 40 or distributed over multiple machines.
WO 2014/044853 38 PCT/EP2013/069751 Having generally described this invention, a further understanding of characteristics and advantages of the invention can be obtained by reference to certain specific examples and figures which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. 5 DESCRIPTION OF THE FIGURES Figure 1: a 55 genes molecular algorithm for the classification and diagnosis of hepatocellular tumors. Sensitivity (sen), specificity (spe), negative predictive value (PNV), positive predictive value (PPV) and accuracy (acc) were detailed underneath each subset of tumors. Genes in each branch of the algorithm were resumed inside the 10 grey boxes. EXAMPLES Example 1. Identification of molecular signatures permitting to classify a liver sample among various types of liver disease Patients and methods 15 Patients and tissue samples Liver samples were systematically frozen following liver resection for tumor in two French University hospitals, in Bordeaux (from 1998 to 2007) and Creteil (From 2003 to 2007). A total of 550 samples were included in this work and the study was approved by the local IRB committee (CCPRB Paris Saint Louis, 1997 and 2004) and all patients 20 gave their informed consent according to French law. Were excluded: (1) tumors with necrosis>80%, (2) tumors with RNA of poor quality or of insufficient amount, (3) HCC with non-curative resection: R1 or R2 resection or extra hepatic metastasis at the time of the surgery, (4) HCC treated by liver transplantation. Accordingly, the following samples were included: 25 0 40 non-hepatocellular tumors, comprising intra-hepatic cholangiocarcinoma (n=19), metastasis of colorectal (n=14) and neuroendocrine (n=2) carcinoma, angiolipoma (n=3), leiomyoma (n=1) and angioma (n=1), * 324 HCC, * 156 benign hepatocellular tumors, including focal nodular hyperplasia (FNH, 30 n=25), hepatocellular adenoma (HCA, n=1 11), regenerative macronodule (with dysplasia, n=15, or without, n=5), and * 30 non-tumor samples, including cirrhosis (n=23 associated to HCV n=10, HBV n=3, alcohol n=7, NASH n=1, primary biliary cirrhosis n=1, alpha-1 antitrypsin deficiency n=1) and 7 normal liver tissues.
WO 2014/044853 39 PCT/EP2013/069751 Molecular subtypes of HCA (B-catenin activated n= 23, HNF1A inactivated n= 26, inflammatory n= 68 and unclassified n= 8) were determined according to the previous molecular classification described in Zucman Rossi J, et al. Hepatology 2006, using gene mutation and immunohistochemistry staining. 14 (12.6 %) HCA exhibited both an 5 inflammatory phenotype and activating mutations of P-catenin. Tumor and non-tumor liver samples were frozen immediately after surgery and conserved at -80'C. Tissue samples from the frozen counterpart were also fixed in 10% formaldehyde, paraffin-embedded and stained with Hematoxylin and Eosin and 10 Masson's trichrome. The diagnosis of HCA, HCC, FNH, macroregenerative nodule and all non-hepatocellular tumors was based on established histological criteria (International working party Hepatology 1995, international consensus group Hepatology 2009). All tumors were assessed independently by 2 expert pathologists (JC and PBS) without knowledge of patient's outcome and initial diagnosis. In case of 15 disagreement regarding the subtype diagnosis of hepatocellular tumors or regarding the pathological features of HCC included in prognosis analysis, sections were re examined and a consensus was reached and used for the study. In the case of multitumors, the largest nodule available was analysed in our prognostic study. Selection of genes for further analysis by quantitative PCR 20 We selected 103 genes for the quantitative RT-PCR analysis. Using Affymetrix HG133A gene chip TM microarray hybridizations performed on the same platform, the mRNA expression of 82 liver samples including 57 HCC (E-TABM-36), 5 HNF1A inactivated adenomas (GSE7473), 7 inflammatory adenomas (GSE11819), 4 focal nodular hyperplasia (GSE9536) 9 non-tumor liver samples including cirrhosis and 25 normal livers (E-TABM-36 and GSE7473) was analyzed. Genes differentially expressed in specific subgroups of tumors were selected according to 3 criteria for inclusion: (1) 38 genes were selected from previous microarray data obtained by the inventors and described in boyault et al and rebouissou JBC Rebouissou 30 Nature and rebouissou J Hepatol: RAB1A, REG3A, NRAS, RAMP3, MERTK, PIR, EPHA1, LAMA3, GOS2, HN1, PAK2, AFP, CYP2C9, CDH2, HAMP, SAE1, NTS, HAL, SDS, cmkORl/CXCR7, ID2, GADD45B, CDT6, UGT2B7, LFABP, GLUL, LGR5/GPR49, TBX3, RHBG, SLPI, AMACR, SAA2, CRP, MME, DHRS2, SLC16A1, GLS2, and GNMT; 35 (2) 9 genes were previously described in the literature (Odom DT, et al. 2004; Paradis V, et al. 2003; Rebouissou S, et al. 2008; Llovet J, et al. 2006; Capurro M, et al. 2003; Chuma M, et al. 2003; Tsunedomi 2005; Kondoh N 1999): HNF1A, HNF4A, SERPIN, ANGPT1, ANGPT2, XLKD1-LYVE1, GPC3, HSP70/HSPA1A, and CYP3A7; and WO 2014/044853 40 PCT/EP2013/069751 (3) 13 genes were selected from new analysis of previous microarray data of the inventors: STEAP3, RRM2, GSN, CYP2C19, C8A, AKR1B10, ESR1, GMNN, CAP2, DPP8, LCAT, NEK7, LAPTM4B. A total of 60 genes were selected for further analysis by quantitative PCR. 5 At this stage, the inventors also wished to provide a new tool for simple and reliable prognosis of HCC, so that further genes found or already described as associated to HCC prognosis were also included for further quantitative PCR analysis: (1) a panel of 41 genes mostly differentially expressed (significance and fold change) between HCC patients characterized by radically different prognosis 10 was identified by new microarray data obtained using Affymetrix microarray E TABM-36 analysis of the pattern of expression of 44 HCC treated by curative resection: TAF9, NRCAM, PSMD1, ARFGEF2, SPP1, CDC20, NRAS, ENO1, RRAGD, CHKA, RAN, TRIP13, IMP-3/IGF2BP3, KLRB1, C14orf156, NPEPPS, PDCD2, PHB, KIAA0090, KPNA2, KIAA0268/UNQ6077/LOC440751, G6PD, 15 STK6, TFRC, GLA, AKR1C1/AKR1C2, GIMAP5, ADM, CCNB1, TKT, AGPS, NUDT9, HLA-DQA1, NEU1, RARRES2, BIRC5, FLJ20273, HMGB3, MPPE1, CCL5, and DLG7; and (2) 2 genes (KRT19 and EPCAM) described in the literature as related to HCC prognosis (Lee JS nat med 2006, Yamashita T gastroenterology 2008). 20 A total of 43 genes were selected for their association with HCC prognosis. Quantitative RT-PCR RNAs extraction and quantitative RT-PCR was performed, as previously described. Expression of the 103 selected genes was analysed in duplicate in all the 550 samples using TaqMan Microfluidic card TLDA (Applied Biosystems) gene expression assays. 25 Gene expression was normalized with the RNA ribosomal 18S, and the level of expression of the tumor sample was compared with the mean level of the corresponding gene expression in normal liver tissues, expressed as an n-fold ratio. The relative amount of RNA was calculated with the 2-delta delta CT method. Mutation screening 30 DNA was extracted and quality was assessed. All HCA samples have been sequenced for CTNNB1 (exon 2 to 4), HNF1A (exon i to 10), IL6ST (exon 6 and 10), GNAS (exon 8) and STAT3 (exon 2, 5 and 20). All HCC samples have been sequenced for CTNNB1 (exon 2 to 4) and TP53 (exons 2 to 11). All mutations were confirmed by sequencing a second independent amplification product on both strands; screening for mutations in 35 the matched non-tumor sample was performed in order to detect any germline mutations. Endpoints for the diagnosis Consensus between pathologists was considered as the gold standard for the diagnosis. We assessed sensitivity (Sen), specificity (Spe), predictive negative value WO 2014/044853 41 PCT/EP2013/069751 (PNV), predictive positive value (PPV) and the accuracy for the diagnosis of HCC, FNH, HCA and the different subtype of HCA. Non-hepatocellular tumors, regenerative macro nodule and non-tumor liver samples (cirrhosis and normal liver) were included in order to assess the ability of the molecular algorithm to distinguish them from HCC, 5 FNH and HCA. The study was not designed to diagnose the specific subtypes of non hepatocellular tumors, the different subtypes of non-tumor liver samples (normal liver and cirrhosis) and of regenerative macronodules. Construction of the molecular diagnostic algorithm The 550 samples were divided into a global training set S1 (n=306) and a global 10 validation set S2 (n=244). This partition was built randomly in order to provide for each variable V to be predicted (hepatocellular type, malignancy, ...) a training set S1v (q S1) and a validation set S 2 v (( S2) both containing approximately 50% of the samples to be analyzed for this variable and with similar proportion of "positive" cases (here all variables are binary, values being either Yes or No; "positives" cases refer to samples 15 taking the value Yes). 103 genes were measured (-AACt measures), and four operators (addition, subtraction, min, max) were applied to all pairs of distinct genes (n=5886) to create new variables, yielding a total of 23653 variables (103 initial, 23544 created). Given a variable V to be predicted the corresponding training set S1v was randomly 20 divided into two subsets S1v.A and S1v.B with equal* size and equal* proportion of "positive" cases (*:or almost equal when n is impair). Then depending on the variable to be predicted (i.e. on the clinical implications) either a criterion giving more weight to Positive Predictive Value (focal nodular hyperplasia, HNF1A, Inflammatory, 0 catenin), or to Sensitivity (hepatocellular, malignancy, 25 adenoma) was chosen. In all cases, the final criterion was obtained as 0.8 criterion 1 4 + 0.2 criterion 2 criterion 1 and criterion 2 corresponding respectively to PPV and sensitivity or conversely). The AUC criteria is then calculated on S1v.A for each of the 23653 variables (PresenceAbsence R package), and the top 2000 variables (ranked by decreasing 30 order of AUC - 2 sd) were then selected for the further steps. A distance matrix between these 2000 variables has then been calculated as 1 pearson correlation coefficient, using S1v.A. A hierarchical clustering has then been performed on this distance matrix and the obtained dendrogram is cut in 50 clusters. In each cluster, the variable yielding the higher value of AUC - 2 sd (obtained at the 35 previous step) was kept. These 50 genes were then used in a stepwise procedure to build multivariate models on Slv. For a given combination of predictive variables, 3 algorithms (DLDA, DQDA, PAM) are trained on S1v.A, yielding 3 predictors, which are then used to predict S1v.B. The criterion is then calculated for each of the 3 predictors independently on S1v.A and 40 S1v.B. Criterion values are then averaged over the 3 predictors and the current model WO 2014/044853 42 PCT/EP2013/069751 was said superior to competitor models if it does as good as them on S1v.A and better on S1v.B. A modified stepwise forward procedure was used: at run k>2 (i.e. building a model at k variables, based on a previously obtained model at (k-1) variables), a variable is 5 added, then a variable is removed and a variable is added again. The variable to be added or removed is selected among those optimizing the criterion. When several variables are optimizing the criterion, the first encountered is selected. 15 models were built, ranging from 1 to 15 genes. The smallest model, i.e. with the less possible variables, optimizing the criterion, was then selected. To validate this model, it was 10 used to predict samples from the validation set S2v. As 3 algorithms are used in the model, a majority rule is used to get a unique class membership. Statistical analysis Continuous and discontinuous variable were compared using Mann Whitney and Chi square or fisher exact test respectively. Univariate and multivariate analysis were 15 performed using the Cox model. Statistical analysis was performed using the R statistical software and rms package. Results A molecular algorithm was constructed for diagnosis as a hierarchic tool used in a 20 decisional tree (see Figure 1). The expression level of all the 103 selected genes was analyzed by quantitative RT PCR. In the overall series of 550 included samples, each subgroup of samples were randomly separated (ratio 1/1) in a training and validation set in order to create and validate the molecular algorithm, respectively. Using a step-by-step analysis, 55 genes 25 have been identified (described in Table 2) that could classify samples in each specific subgroups using a consensus between 3 nearest centroid methods (DLDA, DLQA and PAM, as detailed in Patients and Methods). Then, the robustness of the molecular classifiers was tested in the validation set of tumors (as described in Figure 1 and in Table 3 below). 30 Table 3: accuracy of the molecular algorithm for the diagnosis of hepatocellular tumors among 550 liver samples Training Validation Training + validation Sen Spe PPV NPV Acc Sen Spe PPV NPV Acc Sen Spe PPV NPV Acc n n n (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Non hepatocellular/ 21/285 99.3 94.4 99.7 89.5 99.0 19/225 99.1 100 100 90.5 99.2 40/510 99.2 97.3 99.8 90.0 99.1 Hepatocellular hep t eliar 191/96 97.9 96.8 98.4 95.8 97.6 133/90 98.3 84.8 87.2 97.8 91.5 98.1 90.0 93.8 96.8 94.9 hepatocellular ___ _ _ _ __ ___/186 WO 2014/044853 PCT/EP2013/069751 tissues FNH / others beiHn issuess 13/83 100 100 100 100 100 12/78 100 97.5 83.4 100 97.7 25/161 100 98.8 92.3 100 98.9 H CA / others beni issues 56/37 93.3 100 100 84.7 95.1 55/38 96.5 100 100 91.7 97.5 111/75 94.9 100 100 88 96.3 HNFIA H CA/ 13/43 100 100 100 100 100 13/42 100 100 100 100 100 26/85 100 100 100 100 100 Inflammatory HCA / others 34/22 100 92.3 93.8 100 96.4 34/21 97.2 94.7 97.2 94.7 96.4 68/43 98.5 93.3 95.6 97.7 96.4 HCA* oters HCA 12/44 84.6 95.3 95.3 92.9 95.1 11/44 77.8 93.3 70 95.5 90.7 23/88 81.8 94.3 78.3 95.4 91.8 * 14 (12.6 %) HCA exhibited both an inflammatory phenotype and activating mutations of p catenin Benign hepatocellular tissus (n=186) are composed of FNH (n= 25), HCA (n= 111), normal liver (n=7), cirrhosis (n=23, etiology: HCV n=10, HBV n=3, Alcohol n=7, NASH n=1, primary 5 biliary cirrhosis n=1, alpha-1 antitrypsin deficiency n=1), non-dysplastic regenerative macronodule (n=5) and dysplastic macronodule (n=15). Sen= sensitivity, Spe= specificity, PPV = positive predictive value, NPV= negative predictive value, Acc = accuracy, HCC = hepatocellular carcinoma, FNH = focal nodular hyperplasia, HCA = hepatocellular adenoma 10 First, hepatocellular samples were efficiently identified from non-hepatocellular tumors by combining 9 genes (EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, and C8A, see Figure 1), then, benign hepatocellular samples were discriminated from HCC using a combination of 9 genes (AFP, CAP2, LCAT, ANGPT2, AURKA, CDC20, 15 DHRS2, LYVE1, and ADM, see Figure 1). HCC were also classified using the G1-G6 classification previously described in W02007/063118A1, which permitted to confirm the reliability of this method in a large cohort of HCC, and the relationships previously described with the genetic and clinical features (see Table 4 below). 20 Table 4: Clinical and genetic features associated with G1-G6 classification in HCC included in the diagnostic study (n= 324) Associated Fisher exact test P value group in 324 HCCa Female G1 0.0086 HBV G1-G2 0.0002 Clinical Age < 60 years old G1-G2 0.0001 variables AFP>20 ng/ml G1 <0.0001 Poor prognosis G3 <0.0001 Genetic TP53 mutations G2-G3 <0.0001 alteration CTNNB1 mutations G5-G6 <0.0001 aExcept for prognosis (n=314) WO 2014/044853 PCT/EP2013/069751 Then, focusing on the benign subtypes of hepatocellular tumors, it was possible to identify HCA or FNH from the other benign hepatocellular tissues (including regenerative macronodule, dysplastic macronodule and non-tumor liver tissues) using 13 genes for FNH (HAL, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, 5 RHBG, UGT2B7, LGR5, RARRES2, RBM47, and GIMAP5, see Figure 1) and 13 genes for HCA (HAL, CYP3A7, LCAT, LYVE1, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, see Figure 1). Finally, the different subtypes of HCA we classified: HNF1A mutated (4 genes: FABP1, ANGPT2, DHRS2, and UGT2B7, see Figure 1), P catenin mutated (13 genes: TFRC, 10 HAL, CAP2, GLUL, HMGB3, LGR5, GIMAP5, AKR1B10, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, see Figure 1), and inflammatory adenomas (7 genes: ANGPT2, GLS2, EPHA1, CC15, HAMP, SAA2, and NRCAM, see Figure 1). As shown in Table 3 above, for each type of tumors, more than 90 % were obtained for sensitivity, specificity, negative predictive value, positive predictive value and accuracy 15 in almost each branch of the diagnosis tree in both the training and validation set. These data underline the robustness of the 55 genes classification/diagnosis algorithm according to the invention. Conclusion In this study, a molecular 55-genes algorithm has been identified and validated for the 20 first time to classify both benign and malignant hepatocellular tumors in specific subgroups. In the diagnostic field of hepatocellular tumors, previous study have focused on diagnosis of early HCC, HCA or FNH but they have never captured the whole body of benign and malignant hepatocellular neoplasms (Bioulac Sage P hepatology 2007, Rebouissou S J hepatol 2008, Llovet JM gastroenterology 2006). In 25 difficult cases, the algorithm according to the invention could help the pathological diagnosis by assessing the molecular subclass. The 16 genes of the G1-G6 classification previously described in W02007/063118A1 were also kept in the general algorithm, because different molecular subgroups constitute different potential therapeutic targets (G1 with IGFR1 inhibitor, G1-G2 with 30 mTor inhibitor and G5-G6 with wnt inhibitor) and it could guide future clinical trial. In conclusion, this study constitutes a new step in personalized medicine by providing a classification/diagnosis molecular algorithm to perform a global assessment of liver samples. This may help oncologists to take their therapeutic decisions for patients suspected to suffer from a liver tumor. 35 WO 2014/044853 45 PCT/EP2013/069751 BIBLIOGRAPHIC REFERENCES Benhamouche S, Decaens T, Godard C, et al. Apc tumor suppressor gene is the "zonation-keeper" of mouse liver. Developmental cell 2006;10:759-70. Bioulac-Sage P, Rebouissou S, Thomas C, et al. Hepatocellular adenoma subtype 5 classification using molecular markers and immunohistochemistry. Hepatology 2007;46:740-8. Bioulac-Sage P, Cubel G, Balabaud C, Zucman-Rossi J. Revisiting the pathology of resected benign hepatocellular nodules using new immunohistochemical markers. Seminars in liver disease 2011;31:91-103. 10 Boyault S, Rickman DS, de Reynies A, et al. Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology 2007;45:42-52. Cadoret A, Ovejero C, Terris B, et al. New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 2002;21:8293-301. Capurro M, Wanless IR, Sherman M, et al. Glypican-3: a novel serum and 15 histochemical marker for hepatocellular carcinoma. Gastroenterology 2003;125:89-97. Chuma M, Sakamoto M, Yamazaki K, et al. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology 2003;37:198-207. EI-Serag HB. Hepatocellular carcinoma. The New England journal of medicine 20 2011;365:1118-27. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379:1245-55. Forner A, Vilana R, Ayuso C, et al. Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: Prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma. Hepatology 2008;47:97-104. 25 Terminology of nodular hepatocellular lesions. International Working Party. Hepatology 1995;22:983-93. Pathologic diagnosis of early hepatocellular carcinoma: a report of the international consensus group for hepatocellular neoplasia. Hepatology 2009;49:658-64. Kondoh N, Wakatsuki T, Ryo A, et al. Identification and characterization of genes 30 associated with human hepatocellular carcinogenesis. Cancer research 1999;59:4990 6. Lee JS, Heo J, Libbrecht L, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nature medicine 2006;12:410-6. Llovet JM, Chen Y, Wurmbach E, et al. A molecular signature to discriminate dysplastic 35 nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology 2006;131:1758-67. Odom DT, Zizlsperger N, Gordon DB, et al. Control of pancreas and liver gene expression by HNF transcription factors. Science 2004;303:1378-81. Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and 40 Proteins (Wiley & Sons, Inc., 2 nd ed., 2001) WO 2014/044853 46 PCT/EP2013/069751 Paradis V, Bieche 1, Dargere D, et al. A quantitative gene expression study suggests a role for angiopoietins in focal nodular hyperplasia. Gastroenterology 2003;124:651-9. Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) 5 Rebouissou S, Imbeaud S, Balabaud C, et al. HNFlalpha inactivation promotes lipogenesis in human hepatocellular adenoma independently of SREBP-1 and carbohydrate-response element-binding protein (ChREBP) activation. The Journal of biological chemistry 2007;282:14437-46. Rebouissou S, Couchy G, Libbrecht L, et al. The beta-catenin pathway is activated in 10 focal nodular hyperplasia but not in cirrhotic FNH-like nodules. Journal of hepatology 2008;49:61-71. Rebouissou S, Amessou M, Couchy G, et al. Frequent in-frame somatic deletions activate gpl30 in inflammatory hepatocellular tumours. Nature 2009;457:200-4. Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, 15 Amsterdam, 1998); Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Tsunedomi R, lizuka N, Hamamoto Y, et al. Patterns of expression of cytochrome P450 genes in progression of hepatitis C virus-associated hepatocellular carcinoma. 20 International journal of oncology 2005;27:661-7. van Aalten SM, Verheij J, Terkivatan T, Dwarkasing RS, de Man RA, ljzermans JN. Validation of a liver adenoma classification system in a tertiary referral centre: implications for clinical practice. Journal of hepatology 2011;55:120-5. W02007/063118A1 25 Yamamoto Y, Sakamoto M, Fujii G, et al. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations. Hepatology 2003;37:528-33. Yamashita T, Forgues M, Wang W, et al. EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer research 30 2008;68:1451-61. Zucman-Rossi J, Jeannot E, Nhieu JT, et al. Genotype-phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC. Hepatology 2006;43:515-24.

Claims (16)

1. A method for classifying in vitro a liver sample as a non-hepatocellular sample, a hepatocellular carcinoma (HCC) sample, a focal nodule dysplasia (FNH) 5 sample, a hepatocellular adenoma (HCA) sample or another benign liver sample, comprising: a) Determining in vitro from said liver sample an expression profile comprising or consisting of the 38 following genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, 10 ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and optionally one or more internal control genes,; 15 b) Determining if said liver sample is a hepatocellular or a non hepatocellular sample, based on the expression levels measured for an expression profile comprising or consisting of the 9 following genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, and C8A, and optionally one or more internal control genes, using at least one 20 algorithm calibrated with at least one reference liver sample; c) If said liver sample is a hepatocellular sample, then determining if said hepatocellular sample is a HCC sample or a benign hepatocellular sample, based on the expression levels measured for an expression profile comprising or consisting of the 9 following genes: AFP, CAP2, 25 LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, and ADM, and optionally one or more internal control genes, using at least one algorithm calibrated with at least one reference liver sample; d) If said liver sample is a benign hepatocellular sample, then determining if said benign hepatocellular sample is a FNH sample, based on the 30 expression levels measured for an expression profile comprising or consisting of the 13 following genes: HAL, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, and GIMAP5, and optionally one or more internal control genes, using at least one algorithm calibrated with at least one reference liver sample; 35 e) If said liver sample is a benign hepatocellular sample, then determining if said benign hepatocellular sample is a HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 13 following genes: HAL, CYP3A7, LCAT, LYVE1, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and 40 CYP2C9, and optionally one or more internal control genes, using at least one algorithm calibrated with at least one reference liver sample; WO 2014/044853 48 PCT/EP2013/069751 f) If said benign hepatocellular sample is neither a FNH sample nor a HCA sample, then it is classified as another benign liver sample.
2. The method of claim 1, further comprising, if the liver sample is diagnosed as a 5 HCA sample, classifying said HCA sample into one of the following HCA subgroups: HNF1A mutated HCA, inflammatory HCA, P catenin mutated HCA or other HCA, by: a) Further determining in vitro from said HCA sample an expression profile comprising or consisting of the 8 additional following genes: HAMP, 10 SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3; b) Determining if said HCA sample is or not a HNF1A mutated HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 4 following genes: FABP1, ANGPT2, DHRS2, and UGT2B7, and optionally one or more internal 15 control genes, using at least one algorithm calibrated with at least one reference liver sample; c) Determining if said HCA sample is or not an inflammatory HCA sample, based on the expression levels measured for an expression profile comprising or consisting of the 7 following genes: ANGPT2, GLS2, 20 EPHA1, CC15, HAMP, SAA2, and NRCAM, and optionally one or more internal control genes, using at least one algorithm calibrated with at least one reference liver sample; d) Determining if said HCA sample is or not a P catenin mutated HCA sample, based on the expression levels measured for an expression 25 profile comprising or consisting of the 13 following genes: TFRC, HAL, CAP2, GLUL, HMGB3, LGR5, GIMAP5, AKR1B10, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control genes, using at least one algorithm calibrated with at least one reference liver sample; 30 e) If said HCA sample is neither a HNF1A mutated HCA sample, an inflammatory HCA sample, nor a P catenin mutated HCA sample, then it is classified as another HCA sample.
3. The method according to claim 1 or claim 2, further comprising, if the liver 35 sample is diagnosed as a HCC sample, classifying said HCC sample into one of subgroups G1 to G6 defined by the following clinical and genetic main features: G1 G2 G3 G4 G5 G6 Chromosome instability + + + - - Early relapse and death + + + - - TP53 mutation - + + - - - WO 2014/044853 49 PCT/EP2013/069751 HBV infection + + - - - Low copy number + - - - - High copy number - + - - - CTNNB1 mutation - - - - + + Satellite nodules - - - - - + wherein classification is made by: a) Further determining in vitro from said HCC sample an expression profile comprising or consisting of the 11 additional following genes: RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and 5 SAE1; and b) calculating 6 subgroup distances based on the expression levels measured for an expression profile comprising or consisting of the 16 following genes: RAB1A, REG3A, NRAS, RAMP3, MERTK, PIR, EPHA1, LAMA3, GOS2, HN1, PAK2, AFP, CYP2C9, CDH2, HAMP, and 10 SAE1, and optionally one or more internal control genes; and c) classifying said HCC tumor in the subgroup for which the subgroup distance is the lowest.
4. The method according to any one of claims 1 to 3, wherein reference samples 15 used for calibrating algorithms used for interpreting each expression profile are the following: a) For determining if a liver sample is or not a hepatocellular sample: at least one (preferably several) hepatocellular sample and at least one (preferably several) non-hepatocellular sample; 20 b) For determining if a hepatocellular sample is or not a HCC sample: at least one (preferably several) benign sample and at least one (preferably several) HCC sample; c) For determining if a benign hepatocellular sample is or not a FNH sample: at least one (preferably several) FNH sample and at least one 25 (preferably several) non-FNH benign hepatocellular sample; d) For determining if a benign hepatocellular sample is or not a HCA sample: at least one (preferably several) HCA sample and at least one (preferably several) non-HCA benign hepatocellular sample; e) For determining if a HCA sample is or not a HNF1A mutated HCA 30 sample: at least one (preferably several) HNF1A mutated HCA sample and at least one (preferably several) non-HNF1A mutated HCA sample; f) For determining if a HCA sample is or not an inflammatory HCA sample: at least one (preferably several) inflammatory HCA sample and at least one (preferably several) non-inflammatory HCA sample; 35 g) For determining if a HCA sample is or not a P catenin mutated HCA sample: at least one (preferably several) P catenin mutated HCA sample WO 2014/044853 50 PCT/EP2013/069751 and at least one (preferably several) non-P catenin mutated HCA sample; and h) For classifying a HCC sample into one of subgroups G1 to G6: at least one (preferably several) sample of each G1 to G6 subgroups. 5
5. The method according to any one of claims 1 to 4, wherein said liver sample is a liver biopsy or a partial or whole liver tumor surgical resection.
6. The method according to any one of claims 1 to 5, wherein said expression 10 profile(s) is(are) determined at the nucleic level.
7. The method according to claim 6, wherein said expression profile(s) is(are) determined using quantitative PCR. 15
8. The method according to anyone of claims 1-2 and 4-7, wherein the algorithm(s) used for interpreting any expression profile are selected from: a) Prediction Analysis of Microarrays (PAM): PAM(sample X) = Arg max (yes (sample X); ONo (sample X)) wherein 6 Yes (sample X) = (1 'T X 7TYesji - Kyes i=1 6 No (sample X) = (1 - x N )Noi KNo i=1 wherein: 20 0 xi, 1 i N, represent the in vitro measured values of N variables derived from the expression levels of genes of the expression profile, and e 7T, yi, 7TYes,i, 7TNo,, 1 i N, Kyes and KNo are fixed parameters calibrated with at least one reference sample; 25 b) Diagonal Linear Discriminant Analysis (DLDA): DLDA(sample X) = Arg min(Ayes (sample X); ANo (sample X)) wherein Ayes (sample X) = (xi ANo (sample X) = (Xi - INo,i)2 1 1 wherein: WO 2014/044853 51 PCT/EP2013/069751 * xi, 1 i N, represent the in vitro measured values of N variables derived from the expression levels of genes of the expression profile, and e Ui, pIYes,i, and lINo,i, 1 i N, are fixed parameters calibrated with at 5 least one reference sample; c) Diagonal quadratic discriminant analysis (DQDA): DQDA(sample X) = Arg min (Vyes (sample X); VNo ( sample X)) wherein Vyes (sample X) = X + Cyes S VYesJi VNo(sample X) = (Xi [ 1 No 1)+ CNo wherein: * xi, 1 i N, represent the in vitro measured values of N variables 10 derived from the expression levels of genes of the expression profile, and e UYes,i, NN, Ies,i, INo,i, , 1 i N, are fixed parameters calibrated with at least one reference sample, and Cres = log(VYes,) CNo =1 (ogVNo,i)); 15 d) or any combination thereof.
9. The method of claim 8, wherein the algorithm used for interpreting each expression profile is: Diagnosis (sample X) = majority rule (PAM(sample X), DLDA(sample X), DQDA(sample X)) 20
10. The method according to claim 9, wherein said expression profile(s) is(are) determined using quantitative PCR and the variables and parameters of PAM, DLDA and DQDA algorithms are the following: WO 2014/044853 52 PCT/EP2013/069751 a) For determining if a liver sample is or not a hepatocellular sample: * 6 variables x, to x6 are used as follows: x, (-MACt TFRC expression level) - (-MACt C8A expression level) xz (-MACt AFP expression level) + (-MACt GNMT expression level) x3 (-ACt HAL expression level) - (-MACt EPCAM expression level) x4 (-ACt CYP3A7 expression level) - (-ACt EPCAM expression level) xs (-ACt FABP1 expression level) - (-MACt EPCAM expression level) x6 (-ACt EPCAM expression level) - (-AACt HNF4A expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi 1.342931 -0.09325912 2.006058 7.153821 xz -1.551583 0.10774885 -4.1733248 9.685958 x3 -1.23594 0.08582914 -0.9310016 10.17258 8.151418 0.0932632 x4 -1.524252 0.10585085 2.8897574 10.391148 xs -1.261254 0.08758709 -1.0531553 10.049158 X6 1.087001 -0.07548619 -1.4702021 9.901341 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi UNo,i UYesi Vi Xi 11.613149 1.3388989 11.690171 4.251989 4.692407 xz -19.201897 -3.12967394 12.73627 22.662048 22.074337 x3 -13.503695 -0.05789783 17.965523 27.445047 26.883759 x4 -12.948974 3.98966931 6.765985 30.609874 29.198065 Xs -13.727697 -0.17297876 17.267584 26.144739 25.619118 X6 9.292567 -2.21761661 1.913791 25.543753 24.14461 5 b) For determining if a hepatocellular sample is or not a HCC sample: * 6 variables x, to x6 are used as follows: x, (-ACt CAP2 expression level) - (-ACt LCAT expression level) xz (-ACt ANGPT2 expression level) + (-AACt AURKA expression level) x3 (-ACt CDC20 expression level) + (-MACt DHRS2 expression level) x4 (-ACt ANGPT2 expression level) - (-ACt LYVE1 expression level) xs (-ACt ADM expression level) - (-ACt CDC20 expression level) x6 Max (-ACt AFP expression level; -ACt CAP2 expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes x, -0.16268042 0.08134021 5.787048 4.542418 1.272916 0.449041 x2 -0.22453753 0.11226876 3.035909 3.975872 WO 2014/044853 53 PCT/EP2013/069751 x3 -0.42378458 0.21189229 3.937962 6.248688 x4 -0.2592874 0.1296437 4.151425 3.70769 X5 0.15685585 -0.07842792 -4.403932 3.840179 X6 -0.01726311 0.00863156 3.696066 4.123495 DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi VNo,i VYesi Vi xi 2.678847 7.341149 2.2201 8.37556 6.33819 Xz 0.06943705 4.519144 3.255149 4.0793 3.806517 x3 -1.96933307 6.891609 25.818236 13.894186 17.840878 X4 1.25620635 5.599034 1.863177 3.311281 2.831979 X5 -1.79861246 -5.706591 2.246134 3.814584 3.295449 X6 1.47414444 4.807026 1.020023 6.078697 4.404347 c) For determining if a benign hepatocellular sample is or not a FNH sample: e 12 variables x1 to Xi2 are used as follows: x, Min (-AACt ANGPTL7 expression level; -ACt GLUL expression level) xz (-AACt ANGPT1 expression level) - (-AACt HMGB3 expression level) x3 (-AACt GMNN expression level) + (-AACt RAMP3 expression level) x4 Min (-AACt RHBG expression level; -AACt UGT2B7 expression level) x5 Max (-AACt HAL expression level; -AACt RAMP3 expression level) x6 Min (-AACt LGR5 expression level; -AACt UGT2B7 expression level) x7 (-AACt RAMP3 expression level) + (-ACt UGT2B7 expression level) x8 (-AACt RAMP3 expression level) + (-ACt RARRES2 expression level) x9 Max (-AACt ANGPT1 expression level; -AACt RAMP3 expression level) xo Min (-AACt ANGPT1 expression level; -AACt LGR5 expression level) Xi1 (-ACt RAMP3 expression level) - (-ACt RBM47 expression level) Xi2 Min (-MACt GIMAP5 expression level; -ACt UGT2B7 expression level) 5 e PAM parameters are the following: Xi 7TNo,i TYes,i i Yi KNo KYes x, -0.18469273 1.0817717 -1.72829395 3.243668 xz -0.15724871 0.9210281 0.61243528 2.336453 x3 -0.13637923 0.7987926 1.58326744 2.289755 x4 -0.15358836 0.899589 -3.46104209 3.909901 X5 -0.11234999 0.65805 1.19490255 2.017152 0.2800792 6.1260851 X6 -0.11945816 0.6996835 -2.27683325 3.334501 X7 -0.15338781 0.8984143 -0.04692744 2.922347 X8 -0.14256206 0.8350063 0.60258802 2.277919 X9 -0.11634108 0.6814263 1.54744785 1.913217 WO 2014/044853 54 PCT/EP2013/069751 x10 -0.17351058 1.0162762 -1.4122167 3.581967 x11 -0.15477031 0.9065118 1.45598643 2.048925 X12 -0.07438928 0.4357086 -1.04952428 2.524675 e DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi VNo,i VYesi Vi Xi -2.3273759 1.7806145 4.6402628 0.60826433 4.11435 Xz 0.245031 2.76437457 1.4145492 0.20686229 1.2570248 X3 1.2709924 3.41230679 1.2978397 0.19883833 1.1544917 X4 -4.0615574 0.05626186 8.3471726 0.0196296 7.2609714 Xs 0.9682756 2.52228907 0.6935121 0.30621156 0.6429946 X6 -2.6751666 0.05626186 5.1618051 0.0196296 4.4910865 X7 -0.4951798 2.57855093 3.3012094 0.33314121 2.9140701 X8 0.2778432 2.50466495 1.2384457 0.40087507 1.1291973 X9 1.3248621 2.85116431 0.5424233 0.11837803 0.487113 x10 -2.0337258 2.22805082 6.3954525 0.30614496 5.601195 x11 1.1388737 3.31336105 0.7211325 0.52047864 0.6949603 X12 -1.2373331 0.05049854 1.9692555 0.01620956 1.7145104 d) For determining if a benign hepatocellular sample is or not a HCA sample: e 10 variables x, to xio are used as follows: x, (-MACt AKR1B10 expression level) + (-MACt GLS2 expression level) xz (-ACt LCAT expression level) - (-ACt KRT19 expression level) x3 (-ACt ESR1 expression level) + (-ACt SDS expression level) x4 Max (-ACt MERTK expression level; -ACt LYVE1 expression level) xs Max (-ACt EPHAl expression level; -ACt KRT19 expression level) x6 (-ACt CCL5 expression level) + (-MACt GLS2 expression level) x7 (-ACt HAL expression level) - (-MACt MERTK expression level) x8 (-ACt CYP2C9 expression level) - (-MACt MERTK expression level) x9 (-ACt CCL5 expression level) + (-MACt KRT19 expression level) xo Min (-ACt CYP3A7 expression level; -ACt EPHAl expression level) 5 e PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes xi 1.1300586 -0.52467006 -0.96573089 5.405409 xz -0.6257754 0.29053858 0.10777331 4.174906 x3 -0.583684 0.27099612 1.53413349 3.92968 3.0655113 0.7945744 x4 -0.2101061 0.09754928 0.01545178 2.53848 Xs 0.4031816 -0.18719147 0.76400666 2.906802 X6 0.6342941 -0.29449369 -1.82990856 4.756332 WO 2014/044853 55 PCT/EP2013/069751 X7 0.5211003 -0.24193944 -0.57174662 4.026102 X8 0.3773559 -0.17520095 -0.97286634 3.529012 X9 0.8070427 -0.3746984 -0.75070901 3.946451 x10 0.3875215 -0.17992069 0.02720304 2.927056 e DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi VNo,i VYesi Vi X1 5.142698 -3.8017871 1.9223207 16.202619 11.8086811 Xz -2.5047803 1.3207446 4.8696186 4.8642148 4.8658775 X3 -0.759558 2.5990617 1.5948539 4.8438216 3.8441392 X4 -0.5178985 0.2630787 0.1157701 0.4169368 0.3242701 Xs 1.9359758 0.2198781 0.9741474 0.8373057 0.8794108 X6 1.1870048 -3.2306184 0.5402267 10.9818415 7.769037 X7 1.5262567 -1.5458196 1.0506355 5.6452689 4.2315355 X8 0.358827 -1.5911525 0.2637763 3.3978705 2.4335338 X9 2.4342454 -2.2294378 3.9252834 3.9034702 3.910182 X10 1.1615001 -0.4994349 0.507857 1.1000088 0.9178082 e) For determining if a HCA sample is or not a HNF1A mutated HCA sample: * 2 variables x, to x6 are used as follows: x, (-MACt DHRS2 expression level) - (-MACt UGT2B7 expression level) xz (-MACt ANGPT2 expression level) + (-MACt FABP1 expression level) 5 e PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes X1 -0.2597076 1.817954 -1.130125 6.501417 0.1803095 4.3715711 X2 -0.1615805 1.131063 1.136677 3.83618 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi UNo,i UYesi Vi X1 -2.8185929 10.68915 15.46252 14.3631833 15.343027 X2 0.5168253 5.47564 1.668767 0.7321017 1.566956 f) For determining if a HCA sample is or not an inflammatory HCA sample: e 4 variables x, to x6 are used as follows: x, (-MACt HAMP expression level) + (-MACt SAA2 expression level) xz (-ACt CCL5 expression level) - (-ACt NRCAM expression level) x3 Max (-ACt EPHAl expression level; -ACt KRT19 expression level) x4 (-ACt ANGPT2 expression level) + (-ACt SAA2 expression level) WO 2014/044853 56 PCT/EP2013/069751 PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes x, -0.4760712 0.9521423 4.6430007 6.107883 Xz 0.434627 -0.869254 -0.0574931 5.002872 0.7344381 2.4145044 x3 0.1882468 -0.3764937 1.1521703 3.158128 x4 -0.4549338 0.9098677 4.5882009 4.501345 * DLDA and DQDA parameters are the same, as follows: Xi PNo,i lYesi UNo,i UYesi Vi xi 1.735214 10.4585747 16.9585649 7.6603747 13.9265464 Xz 2.11689 -4.4062595 7.0569419 6.5761749 6.90017 X3 1.746678 -0.0368447 0.7298408 0.3673544 0.6116387 X4 2.540387 8.6838292 4.4787841 4.5955546 4.5168614 g) For determining if a HCA sample is or not a P catenin mutated HCA sample: 5 * 9 variables x, to x6 are used as follows: x, (-MACt AKR1B1O expression level) - (-MACt REG3A expression level) x2 (-MACt AMACR expression level) + (-MACt HAL expression level) x3 (-ACt CAP2 expression level) - (-ACt GLUL expression level) x4 (-ACt HAL expression level) + (-ACt TAF9 expression level) xs (-ACt CAP2 expression level) - (-ACt LGR5 expression level) x6 Min (-ACt AKR1B1O expression level; -ACt HAL expression level) x7 (-ACt LAPTM4B expression level) + (-ACt TFRC expression level) x8 (-ACt GIMAP5 expression level) - (-ACt HAL expression level) x9 (-ACt HMGB3 expression level) - (-ACt IGF2BP3 expression level) * PAM parameters are the following: Xi 7TNo,i TYesi i Yi KNo KYes x, 0.34708654 -1.9668237 1.94438201 7.392962 xz 0.21863143 -1.2389115 -1.04516656 3.127947 x3 0.18579207 -1.0528217 1.22379671 2.663529 x4 0.24406366 -1.3830274 0.05214403 3.244264 Xs 0.15694722 -0.8893676 2.7521494 3.869139 0.3607787 8.2634614 X6 0.21470021 -1.2166345 -1.47714108 4.260375 x7 0.11140632 -0.6313025 0.81968112 3.203963 X8 -0.22080529 1.25123 0.49103172 3.193991 X9 0.04764503 -0.2699885 0.56180483 3.025541 WO 2014/044853 57 PCT/EP2013/069751 DLDA and DQDA parameters are the same, as follows: Xi PNo,i PYesi VNo,i VYesi Vi Xi 4.5103796 -12.5962709 37.671414 6.2381109 33.535453 Xz -0.361299 -4.920416 1.426277 8.2837077 2.328571 X3 1.7186592 -1.5804241 1.203395 0.6218992 1.126882 X4 0.8439509 -4.4347616 1.358794 11.5298442 2.69709 Xs 3.3594 -0.6889375 5.646265 1.7986761 5.140003 X6 -0.5624378 -6.6604599 6.819184 8.7029888 7.067053 X7 1.1766229 -1.2029889 2.912529 0.2815287 2.566345 X8 -0.2142184 4.4874493 1.580383 8.8316336 2.534495 X9 0.7059568 -0.2550566 2.287403 0.3047094 2.026522
11. The method according to anyone of claims 3-10, wherein the HCC sample is classified into one of subgroups G1 to G6 using the following formula for 5 calculating the distance of said HCC sample to each subgroup Gk, 1 5k56: Distance (HCC sample, subgroup Gk) = (ACt (HCC sample,subgroup Gk,genet) - p (subgroup Gk, genet)) 2 t=1..16 o(genet) wherein for each genet and subgroup Gk, the p(subgroup Gk, genet) and o-(genet) values are the following: 10 G1 G2 G3 G4 G5 G6 o gene 1 (RABIA) -16.39 -16.04 -16.29 -17.15 -17.33 -16.95 0.23 gene 2 (PAP) -28.75 -27.02 -23.48 -27.87 -19.23 -11.33 16.63 gene 3 (NRAS) -16.92 -17.41 -16.25 -17.31 -16.96 -17.26 0.27 gene 4 (RAMP3) -23.54 -23.12 -25.34 -22.36 -23.09 -23.06 1.23 gene 5 (MERTK) -18.72 -18.43 -21.24 -18.29 -17.03 -16.16 7.23 gene 6 (PIR) -18.44 -19.81 -16.73 -18.28 -17.09 -17.25 0.48 gene 7 (EPHA1) -16.68 -16.51 -19.89 -17.04 -18.70 -21.98 1.57 gene 8 (LAMA3) -20.58 -20.44 -20.19 -21.99 -18.77 -16.85 2.55 gene 9 (GOS2) -14.82 -17.45 -18.18 -14.78 -17.99 -16.06 3.88 gene 10 (HNI) -16.92 -17.16 -15.91 -17.88 -17.72 -17.93 0.54 gene 11 (PAK2) -17.86 -16.56 -16.99 -18.14 -17.92 -17.97 0.58 gene 12 (AFP) -16.68 -12.36 -26.80 -27.28 -25.97 -23.47 14.80 gene 13 (CYP2C9) -18.27 -16.99 -16.26 -16.23 -13.27 -14.44 5.47 gene 14 (CDH2) -15.20 -14.76 -18.91 -15.60 -15.48 -17.32 10.59 gene 15 (HAMP) -19.53 -20.19 -21.32 -18.51 -25.06 -26.10 13.08 gene 16 (SAEI) -17.37 -17.10 -16.79 -18.22 -17.72 -18.16 0.31 WO 2014/044853 58 PCT/EP2013/069751
12. A kit comprising reagents for the determination of an expression profile comprising at most 65 distinct genes, wherein said expression profile is selected from: " An expression profile comprising or consisting of the following 38 genes: 5 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and CYP2C9, and 10 optionally one or more internal control genes; * An expression profile comprising or consisting of the following 46 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, 15 UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control genes; * An expression profile comprising or consisting of the following 49 genes: 20 EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, RAB1A, 25 REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and SAE1, and optionally one or more internal control genes; or * An expression profile comprising or consisting of the following 55 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, 30 ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, IGF2BP3, RAB1A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, and SAE1, and optionally one 35 or more internal control genes. WO 2014/044853 59 PCT/EP2013/069751
13. The kit according to claim 12, comprising: a) specific amplification primers pairs and/or probes, or b) a nucleic acid microarray. 5
14. An IGFR1 inhibitor, an Akt/mTor inhibitor, a proteasome inhibitor and/or a wnt inhibitor, for use in the treatment of HCC in a subject that has been diagnosed as suffering from HCC based on a liver sample that has been classified as a HCC sample by the classification method according to any one of claims 1 to 11. 10
15. A system 1 for classifying a liver sample comprising: a) a determination module 2 configured to receive a liver sample and to determine expression level information concerning: * An expression profile comprising or consisting of the following 38 15 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, and 20 CYP2C9, and optionally one or more internal control genes; * An expression profile comprising or consisting of the following 46 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, 25 RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, LAPTM4B, and IGF2BP3, and optionally one or more internal control genes; 30 0 An expression profile comprising or consisting of the following 49 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, 35 AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, RAB1A, REG3A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, HAMP, and SAE1, and optionally one or more internal control genes; or * An expression profile comprising or consisting of the following 55 40 genes: EPCAM, HNF4A, CYP3A7, FABP1, HAL, AFP, GNMT, TFRC, C8A, CAP2, LCAT, ANGPT2, AURKA, CDC20, DHRS2, WO 2014/044853 60 PCT/EP2013/069751 LYVE1, ADM, ANGPTL7, GLUL, ANGPT1, HMGB3, GMNN, RAMP3, RHBG, UGT2B7, LGR5, RARRES2, RBM47, GIMAP5, AKR1B10, GLS2, KRT19, ESR1, SDS, MERTK, EPHA1, CCL5, CYP2C9, HAMP, SAA2, NRCAM, REG3A, AMACR, TAF9, 5 LAPTM4B, IGF2BP3, RAB1A, NRAS, PIR, LAMA3, GOS2, HN1, PAK2, CDH2, and SAE1, and optionally one or more internal control genes. b) a storage device 3 configured to store the expression level information from the determination module; 10 c) a comparison module 4, adapted to compare the expression level information stored on the storage device with reference data, and to provide a comparison result, wherein the comparison result is indicative of the type of liver sample; and d) a display module 5 for displaying a content 6 based in part on the 15 classification result for the user, wherein the content is a signal indicative of the type of liver sample.
16. A computer readable medium 7 having computer readable instructions recorded thereon to define software modules for implementing on a computer steps of a 20 prognosis method according to anyone of claims 1 to 11 relating to interpretation of expression profiles data.
AU2013320165A 2012-09-21 2013-09-23 A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma Abandoned AU2013320165A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201261704383P 2012-09-21 2012-09-21
EP12306145 2012-09-21
EP12306145.9 2012-09-21
US61/704,383 2012-09-21
PCT/EP2013/069751 WO2014044853A1 (en) 2012-09-21 2013-09-23 A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma

Publications (1)

Publication Number Publication Date
AU2013320165A1 true AU2013320165A1 (en) 2015-04-02

Family

ID=47049104

Family Applications (1)

Application Number Title Priority Date Filing Date
AU2013320165A Abandoned AU2013320165A1 (en) 2012-09-21 2013-09-23 A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma

Country Status (8)

Country Link
US (1) US20150299798A1 (en)
EP (1) EP2898092A1 (en)
JP (1) JP2016500512A (en)
CN (1) CN104755627A (en)
AU (1) AU2013320165A1 (en)
BR (1) BR112015006302A2 (en)
CA (1) CA2884455A1 (en)
WO (1) WO2014044853A1 (en)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016094457A1 (en) * 2014-12-08 2016-06-16 Ray Partha S Methods for treating basal-like and claudin-low breast cancer and combination therapies thereof
EP3193901B1 (en) * 2014-12-23 2018-04-04 4D Pharma Research Limited Pirin polypeptide and immune modulation
CN105079821A (en) * 2015-06-11 2015-11-25 中国人民解放军第二军医大学 Application of long noncoding RNA HNF1A-AS1 ((hepatocyte nuclear factor-1Alpha Antisense 1) in preparation of drugs for treating human malignant solid tumors
EP3326095B1 (en) * 2015-07-17 2024-02-07 Life Technologies Corporation Tool for visualizing pcr results
WO2017011929A1 (en) * 2015-07-17 2017-01-26 北京大学第一医院 Use of substance detecting content of angiopoietin-like protein 2 in serum for preparing products for detecting inflammation and degree of fibrosis of liver
CN108333366B (en) * 2018-01-26 2020-06-12 南通大学附属医院 Method for establishing experimental monitoring rat model for malignant transformation process of liver cells
CN108179192A (en) * 2018-02-06 2018-06-19 徐州医科大学 A kind of kit of gene pleiomorphism variant sites early diagnosis carcinoma of endometrium
AU2020210630B2 (en) 2019-01-23 2025-10-23 Regeneron Pharmaceuticals, Inc. Treatment of ophthalmic conditions with angiopoietin-like 7 (ANGPTL7) inhibitors
US11845989B2 (en) 2019-01-23 2023-12-19 Regeneron Pharmaceuticals, Inc. Treatment of ophthalmic conditions with angiopoietin-like 7 (ANGPTL7) inhibitors
EP3935581A4 (en) 2019-03-04 2022-11-30 Iocurrents, Inc. Data compression and communication using machine learning
CN109758577A (en) * 2019-03-15 2019-05-17 中国科学院上海高等研究院 Use of DHRS2 gene and its inhibitor in preparing medicine for treating liver cancer
CN111458509B (en) * 2020-04-14 2023-09-22 中国人民解放军海军军医大学第三附属医院 Biomarkers for prognostic assessment of hepatocellular carcinoma and kits and methods thereof
CN112501299A (en) * 2020-12-08 2021-03-16 赵景民 Method for predicting recurrence and metastasis of liver cancer and application
EP4298219A1 (en) 2021-02-26 2024-01-03 Regeneron Pharmaceuticals, Inc. Treatment of inflammation with glucocorticoids and angiopoietin-like 7 (angptl7) inhibitors
CN116895333B (en) * 2023-07-06 2025-04-11 南通大学 A prognostic model of lysosome-related genes in hepatocellular carcinoma and its construction method and application
CN117604108B (en) * 2024-01-23 2024-04-09 杭州华得森生物技术有限公司 Biomarker for liver cancer diagnosis and prognosis and application thereof
CN120427356B (en) * 2025-07-08 2025-09-02 杭州华得森生物技术有限公司 Method and kit for enriching liver cancer circulating tumor cells by utilizing GPC3 and MERTK

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1830289A1 (en) 2005-11-30 2007-09-05 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods for hepatocellular carninoma classification and prognosis
SG183067A1 (en) * 2007-07-20 2012-08-30 Cellartis Ab A novel population of hepatocytes derived via definitive endoderm (de-hep) from human blastocysts derived stem cells
US8168390B2 (en) * 2009-05-27 2012-05-01 University Of Regensburg Method and apparatus for diagnosing age-related macular degeneration

Also Published As

Publication number Publication date
CA2884455A1 (en) 2014-03-27
US20150299798A1 (en) 2015-10-22
JP2016500512A (en) 2016-01-14
EP2898092A1 (en) 2015-07-29
WO2014044853A1 (en) 2014-03-27
WO2014044853A8 (en) 2015-06-04
BR112015006302A2 (en) 2017-07-04
CN104755627A (en) 2015-07-01

Similar Documents

Publication Publication Date Title
AU2013320165A1 (en) A new method for classification of liver samples and diagnosis of focal nodule dysplasia, hepatocellular adenoma, and hepatocellular carcinoma
Filella et al. Emerging biomarkers in the diagnosis of prostate cancer
van der Heijden et al. A five-gene expression signature to predict progression in T1G3 bladder cancer
Thangaraj et al. Molecular portrait of oral tongue squamous cell carcinoma shown by integrative meta-analysis of expression profiles with validations
Zhang et al. Circulating microRNA expressions in colorectal cancer as predictors of response to chemotherapy
Riester et al. Combination of a novel gene expression signature with a clinical nomogram improves the prediction of survival in high-risk bladder cancer
Yuan et al. Overexpression of COL3A1 confers a poor prognosis in human bladder cancer identified by co-expression analysis
Khodadadi-Jamayran et al. Prognostic role of elevated mir-24-3p in breast cancer and its association with the metastatic process
US20150232944A1 (en) Method for prognosis of global survival and survival without relapse in hepatocellular carcinoma
Liu et al. Circular RNA profiling identified as a biomarker for predicting the efficacy of Gefitinib therapy for non-small cell lung cancer
WO2012151212A1 (en) Multifocal hepatocellular carcinoma microrna expression patterns and uses thereof
Molinari et al. Biomarkers and molecular imaging as predictors of response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer
WO2015073949A1 (en) Method of subtyping high-grade bladder cancer and uses thereof
CN113430266A (en) Application of G6PC and genome thereof in preparation of renal clear cell carcinoma diagnosis or prognosis evaluation system
Wang et al. Identification of a 5-gene signature for clinical and prognostic prediction in gastric cancer patients upon microarray data
CN113462776A (en) m6Application of A modification-related combined genome in prediction of immunotherapy efficacy of renal clear cell carcinoma patient
US9790557B2 (en) Methods and systems for determining a likelihood of adverse prostate cancer pathology
Thakore et al. Up-regulation of extracellular-matrix and inflammation related genes in oral squamous cell carcinoma
Dang et al. NELFE-dependent MYC signature identifies a unique cancer subtype in hepatocellular carcinoma
Yamanaka et al. Circulating and tissue miR-100 acts as a potential diagnostic biomarker for cervical cancer
Wang et al. UriBLAD: A urine-based gene expression assay for noninvasive detection of bladder cancer
WO2018049506A1 (en) Mirna prostate cancer marker
US20250051848A1 (en) High-accuracy prediction of colorectal cancer chemotherapy efficacy using machine learning applied to gene expression data
WO2017193062A1 (en) Gene signatures for renal cancer prognosis
US20240093306A1 (en) Micro rna liver cancer markers and uses thereof

Legal Events

Date Code Title Description
MK1 Application lapsed section 142(2)(a) - no request for examination in relevant period